U.S. patent application number 16/318793 was filed with the patent office on 2019-08-29 for reagent compounds, compositions, kits, and methods for amplified assays.
This patent application is currently assigned to CELL IDX, INC.. The applicant listed for this patent is CELL IDX, INC., THE UNIVERSITY OF CHICAGO. Invention is credited to Stephen J. KRON, David A. SCHWARTZ.
Application Number | 20190265235 16/318793 |
Document ID | / |
Family ID | 60992500 |
Filed Date | 2019-08-29 |
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United States Patent
Application |
20190265235 |
Kind Code |
A1 |
SCHWARTZ; David A. ; et
al. |
August 29, 2019 |
REAGENT COMPOUNDS, COMPOSITIONS, KITS, AND METHODS FOR AMPLIFIED
ASSAYS
Abstract
The instant disclosure provides reagent compounds, and antibody
and oligonucleotide reagents, for use in a variety of assays,
including immunoassays and nucleic acid hybridizations. The reagent
compounds comprise a bridging antigen or bridging oligonucleotide
and a latent crosslinker moiety, such as a tyramide moiety. The
bridging antigens are recognizable by the antibody of a
corresponding antibody reagent with high affinity, and the bridging
oligonucleotides are complementary to the oligonucleotide of a
corresponding oligonucleotide reagent. The antibody reagents and
oligonucleotide reagents also comprise a crosslinker activation
agent, such as a peroxidase enzyme. Reaction of the reagent
compounds with the crosslinker activation agent results in the
amplification of signal in assays for target cellular markers,
including cellular antigens and nucleic acids. Also provided are
detectable antibodies specific for the bridging antigens, kits
comprising the reagent compounds and antibody and oligonucleotide
reagents, methods of signal amplification using the compounds and
reagents of the disclosure, methods of preparation of the compounds
and reagents, and compositions comprising the compounds and
reagents.
Inventors: |
SCHWARTZ; David A.;
(Encinitas, CA) ; KRON; Stephen J.; (Oak Park,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELL IDX, INC.
THE UNIVERSITY OF CHICAGO |
San Diego
Chicago |
CA
IL |
US
US |
|
|
Assignee: |
CELL IDX, INC.
San Diego
CA
THE UNIVERSITY OF CHICAGO
Chicago
IL
|
Family ID: |
60992500 |
Appl. No.: |
16/318793 |
Filed: |
July 18, 2017 |
PCT Filed: |
July 18, 2017 |
PCT NO: |
PCT/US2017/042656 |
371 Date: |
January 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62363821 |
Jul 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/535 20130101;
C12Q 2543/101 20130101; C12Y 101/03004 20130101; C12N 9/0065
20130101; C12Q 2531/101 20130101; C12Q 1/6804 20130101; C12N 9/0006
20130101; G01N 33/547 20130101; C12Y 301/03001 20130101; C12Y
111/01007 20130101; C12Q 1/682 20130101; C12Q 1/6804 20130101; G01N
33/54353 20130101; C12Q 1/682 20130101; C12N 9/16 20130101; G01N
33/533 20130101; C12Q 2543/101 20130101; C12Q 2531/101 20130101;
C12Q 2531/125 20130101; C12Q 2531/125 20130101 |
International
Class: |
G01N 33/547 20060101
G01N033/547; G01N 33/535 20060101 G01N033/535; G01N 33/533 20060101
G01N033/533; C12N 9/08 20060101 C12N009/08; C12N 9/16 20060101
C12N009/16; C12N 9/04 20060101 C12N009/04 |
Claims
1. A reagent compound comprising: a bridging antigen and a latent
crosslinker moiety, wherein the bridging antigen is not a biotin, a
hapten, or an antigenic fluorophore.
2. The reagent compound of claim 1, wherein the bridging antigen
comprises a polymer.
3. The reagent compound of claim 1, wherein the bridging antigen
comprises a peptide.
4. The reagent compound of claim 1, wherein the bridging antigen
comprises a plurality of antigenic determinants.
5. The reagent compound of claim 4, wherein each antigenic
determinant in the plurality of antigenic determinants is the
same.
6. The reagent compound of claim 4, wherein the plurality of
antigenic determinants comprises a linear repeating structure.
7. The reagent compound of claim 6, wherein the linear repeating
structure comprises a linear repeating peptide structure.
8. The reagent compound of claim 4, wherein the plurality of
antigenic determinants comprises at least three antigenic
determinants.
9. The reagent compound of claim 4, wherein the bridging antigen
comprises a branched structure.
10. The reagent compound of claim 1, wherein the bridging antigen
comprises a peptide comprising a non-natural residue.
11. The reagent compound of claim 10, wherein the non-natural
residue is a non-natural stereoisomer.
12. The reagent compound of claim 10, wherein the non-natural
residue is a .beta.-amino acid.
13. The reagent compound of claim 1, wherein the bridging antigen
and the latent crosslinker moiety are linked by a chemical coupling
reaction through a conjugation moiety.
14. The reagent compound of claim 1, wherein the latent crosslinker
moiety comprises a phenol moiety.
15. The reagent compound of claim 14, wherein the latent
crosslinker moiety comprises a tyramine, a tyramide, or a
tyrosine.
16. An antibody reagent comprising: a crosslinker activation agent
and an antibody specific for a bridging antigen with high
affinity.
17. The antibody reagent of claim 16, wherein the crosslinker
activation agent comprises an enzyme.
18. The antibody reagent of claim 17, wherein the enzyme is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
19. The antibody reagent of claim 18, wherein the enzyme is a
peroxidase.
20. The antibody reagent of claim 19, wherein the peroxidase is a
horseradish peroxidase or a soybean peroxidase.
21. The antibody reagent of claim 16, wherein the antibody is
specific for a bridging antigen comprising a peptide.
22. The antibody reagent of claim 16, wherein the antibody is
specific for a bridging antigen comprising a plurality of antigenic
determinants.
23. The antibody reagent of claim 22, wherein each antigenic
determinant in the plurality of antigenic determinants is the
same.
24. The antibody reagent of claim 22, wherein the plurality of
antigenic determinants comprises a linear repeating structure.
25. The antibody reagent of claim 24, wherein the linear repeating
structure comprises a linear repeating peptide structure.
26. The antibody reagent of claim 22, wherein the plurality of
antigenic determinants comprises at least three antigenic
determinants.
27. The antibody reagent of claim 22, wherein the bridging antigen
comprises a branched structure.
28. The antibody reagent of claim 16, wherein the antibody is
specific for a bridging antigen comprising a peptide comprising a
non-natural residue.
29. The antibody reagent of claim 28, wherein the non-natural
residue is a non-natural stereoisomer.
30. The antibody reagent of claim 28, wherein the non-natural
residue is a .beta.-amino acid.
31. The antibody reagent of claim 16, wherein the antibody is
specific for the bridging antigen with a dissociation constant of
at most 1 nM.
32. The antibody reagent of claim 16, wherein the crosslinker
activation agent and the antibody are linked by a chemical coupling
reaction through a conjugation moiety.
33. The antibody reagent of claim 16, wherein the antibody reagent
comprises added phenol moieties.
34. The antibody reagent of claim 33, wherein the added phenol
moieties are added tyrosine moieties.
35. The antibody reagent of claim 34, wherein the added tyrosine
moieties are residues in a peptide coupled to the antibody
reagent.
36. A detectable antibody comprising: an antibody specific for a
bridging antigen with high affinity and a detectable label.
37. A diagnostic kit comprising: a first reagent compound
comprising a bridging antigen and a latent crosslinker moiety; a
first detectable antibody comprising an antibody specific for the
bridging antigen with high affinity; and instructions for use.
38. The kit of claim 37, wherein the bridging antigen of the first
reagent compound is not a biotin, a hapten, or an antigenic
fluorophore.
39. The kit of claim 37, wherein the bridging antigen of the first
reagent compound comprises a polymer.
40. The kit of claim 37, wherein the bridging antigen of the first
reagent compound comprises a peptide.
41. The kit of claim 37, wherein the first antibody reagent is
specific for the bridging antigen with a dissociation constant of
at most 1 nM.
42. The kit of claim 37, wherein the first detectable antibody
comprises a detectable label.
43. The kit of claim 42, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
44. The kit of claim 43, wherein the detectable label is a
fluorophore.
45. The kit of claim 42, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
46. The kit of claim 37, further comprising a first antibody
reagent; wherein the first antibody reagent comprises an antibody
and a crosslinker activation agent.
47. The kit of claim 46, wherein the crosslinker activation agent
comprises an enzyme.
48. The kit of claim 47, wherein the enzyme is a peroxidase, an
alkaline phosphatase, or a glucose oxidase.
49. The kit of claim 48, wherein the enzyme is a peroxidase.
50. The kit of claim 49, wherein the peroxidase is a horseradish
peroxidase or a soybean peroxidase.
51. The kit of claim 46, wherein the antibody of the first antibody
reagent is specific for a bridging antigen with high affinity.
52. The kit of claim 46, wherein the antibody of the first antibody
reagent is specific for a cellular marker.
53. The kit of claim 52, wherein the cellular marker is selected
from the group consisting of: 4-1BB, AFP, ALK1, Amyloid A, Amyloid
P, Androgen Receptor, Annexin A1, ASMA, BCA225, BCL-1, BCL-2,
BCL-6, BerEP4, Beta-Catenin, Beta-HCG, BG-8, BOB-1, CA19-9, CA125,
Calcitonin, Caldesmon, Calponin-1, Calretinin, CAM 5.2, CD1a, CD2,
CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD21, CD22, CD23,
CD25, CD30, CD31, CD33, CD34, CD38, CD42b, CD43, CD45 LCA, CD45RO,
CD47, CD56, CD57, CD61, CD68, CD79a, CD80, CD86, CD99, CD117,
CD138, CD163, CDX2, CEA, Chromogranin A, CMV, c-kit, c-MET, c-MYC,
Collagen Type IV, Complement 3c (C3c), COX-2, CXCR5, CK1, CK5, CK6,
CK7, CK8, CK14, CK18, CK17, CK19, CK20, CK903, CK AE1, CK AE1/AE3,
CSF-1, CSF-1R, D2-40, Desmin, DOG-1, E-Cadherin, EGFR, EMA, ER,
ERCC1, Factor VIII-RA, Factor XIIIa, Fascin, FoxP1, FoxP3,
Galectin-3, GATA-3, GATA-4, GCDFP-15, GCET1, GFAP, GITR,
Glycophorin A, Glypican 3, Granzyme B, HBME-1, Helicobacter pylori,
Hemoglobin A, Hep Par 1, HER2, HHV-8, HMB-45, HSV l/ll, ICOS,
IFNgamma, IgA, IgD, IgG, IgM, IL17, IL4, Inhibin, iNOS, Kappa Ig
Light Chain, Ki-67, LAG-3, Lambda Ig Light Chain, Lysozyme,
Mammaglobin A, MART-1/Melan A, Mast Cell Tryptase, MHC Class II,
MLH1, MOC-31, MPO, MSA, MSH2, MSH6, MUC1, MUC2, MUM1, MyoD1,
Myogenin, Myoglobin, Napsin A, Nestin, NSE, Oct-2, OX40, OX40L,
p16, p21, p27, p40, p53, p63, p504s, PAX-5, PAX-8, PD-1, PD-L1,
Perforin, PHH3, PIN-4, PLAP, PMS2, Pneumocystis jiroveci (carinii),
PR, PSA, PSAP, RCC, S-100, SMA, SMM, Smoothelin, SOX10, SOX11,
Surfactant Apoprotein A, Synaptophysin, TAG 72, T-bet, TdT,
Thrombomodulin, Thyroglobulin, TIA-1, TIM3, TRAcP, TTF-1,
Tyrosinase, Uroplakin, VEGF, VEGFR-2, Villin, Vimentin, and
WT-1.
54. The kit of claim 46, wherein the antibody of the first antibody
reagent is specific for a cross-species immunoglobulin.
55. The kit of claim 46, wherein the first antibody reagent
comprises added phenol moieties.
56. The kit of claim 55, wherein the added phenol moieties are
added tyrosine moieties.
57. The kit of claim 56, wherein the added tyrosine moieties are
residues in a peptide coupled to the first antibody reagent.
58. The kit of claim 37, further comprising a second detectable
antibody and a second reagent compound.
59. The kit of claim 58, further comprising a first antibody
reagent and a second antibody reagent; wherein each antibody
reagent comprises an antibody and a crosslinker activation
agent.
60. A method for signal amplification comprising: providing a first
sample comprising a first target antigen; reacting the first target
antigen with a first antibody reagent, wherein the first antibody
reagent comprises an antibody specific for the first target antigen
and a crosslinker activation agent; reacting the first antibody
reagent with a first reagent compound, wherein the first reagent
compound comprises a bridging antigen and a latent crosslinker
moiety; and reacting the bridging antigen with a first detectable
antibody comprising an antibody specific for the bridging antigen
with high affinity.
61. The method of claim 60, wherein the crosslinker activation
agent of the first antibody reagent comprises an enzyme.
62. The method of claim 61, wherein the enzyme is a peroxidase, an
alkaline phosphatase, or a glucose oxidase.
63. The method of claim 62, wherein the enzyme is a peroxidase.
64. The method of claim 63, wherein the peroxidase is a horseradish
peroxidase or a soybean peroxidase.
65. The method of claim 60, wherein the first target antigen is a
cellular marker.
66. The method of claim 60, wherein the first target antigen is a
primary antibody specific for a cellular marker.
67. The method of claim 66, wherein the primary antibody is from a
different species than the antibody of the first antibody
reagent.
68. The method of claim 60, wherein the first target antigen is a
bridging antigen.
69. The method of claim 68, wherein the first antibody reagent is
specific for the bridging antigen with a dissociation constant of
at most 1 nM.
70. The method of claim 60 wherein the bridging antigen of the
first reagent compound is not a biotin, a hapten, or an antigenic
fluorophore.
71. The method of claim 60, wherein the first detectable antibody
comprises a detectable label.
72. The method of claim 71, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
73. The method of claim 72, wherein the detectable label is a
fluorophore.
74. The method of claim 72, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
75. The method of claim 70, further comprising: detecting the first
detectable antibody.
76. A method for signal amplification comprising: providing a first
sample comprising a first target antigen; reacting the first target
antigen with a first antibody reagent, wherein the first antibody
reagent comprises an antibody specific for the first target antigen
and a crosslinker activation agent; reacting the first antibody
reagent with a first reagent compound, wherein the first reagent
compound comprises a bridging antigen and a latent crosslinker
moiety; reacting the bridging antigen of the first reagent compound
with a second antibody reagent, wherein the second antibody reagent
comprises an antibody specific for the bridging antigen and a
crosslinker activation agent; and reacting the second antibody
reagent with a second reagent compound, wherein the second reagent
compound comprises a bridging antigen and a latent crosslinker
moiety.
77. The method of claim 76, wherein the crosslinker activation
agent of the second antibody reagent comprises an enzyme.
78. The method of claim 77, wherein the enzyme is a peroxidase, an
alkaline phosphatase, or a glucose oxidase.
79. The method of claim 78, wherein the enzyme is a peroxidase.
80. The method of claim 79, wherein the peroxidase is a horseradish
peroxidase or a soybean peroxidase.
81. The method of claim 76, wherein the first target antigen is a
cellular marker.
82. The method of claim 76, wherein the first target antigen is a
primary antibody specific for a cellular marker.
83. The method of claim 82, wherein the primary antibody is from a
different species than the antibody of the first antibody
reagent.
84. The method of claim 76, wherein the second antibody reagent is
specific for the bridging antigen of the first reagent compound
with a dissociation constant of at most 1 nM.
85. The method of claim 76, wherein the bridging antigen of the
first reagent compound and the bridging antigen of the second
reagent compound are the same.
86. The method of claim 76, further comprising: reacting the
bridging antigen of the second reagent compound with a first
detectable antibody specific for the bridging antigen with high
affinity.
87. The method of claim 86, wherein the first detectable antibody
comprises a detectable label.
88. The method of claim 87, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
89. The method of claim 88, wherein the detectable label is a
fluorophore.
90. The method of claim 88, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
91. The method of claim 86, further comprising: detecting the first
detectable antibody.
92. A reagent composition comprising: an antibody reagent
comprising a crosslinker activation agent and an antibody; and a
reagent compound of any one of claims 1-15.
93. The reagent composition of claim 92, wherein the crosslinker
activation agent comprises an enzyme.
94. The reagent composition of claim 93, wherein the enzyme is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
95. The reagent composition of claim 94, wherein the enzyme is a
peroxidase.
96. The reagent composition of claim 95, wherein the peroxidase is
a horseradish peroxidase or a soybean peroxidase.
97. The reagent composition of claim 92, wherein the antibody
reagent is specific for a bridging antigen with high affinity.
98. The reagent composition of claim 97, wherein the antibody
reagent is specific for a bridging antigen comprising a
peptide.
99. The reagent composition of claim 97, wherein the antibody
reagent is specific for a bridging antigen comprising a plurality
of antigenic determinants.
100. The reagent composition of claim 99, wherein each antigenic
determinant in the plurality of antigenic determinants is the
same.
101. The reagent composition of claim 99, wherein the plurality of
antigenic determinants comprises a linear repeating structure.
102. The reagent composition of claim 101, wherein the linear
repeating structure comprises a linear repeating peptide
structure.
103. The reagent composition of claim 99, wherein the plurality of
antigenic determinants comprises at least three antigenic
determinants.
104. The reagent composition of claim 97, wherein the antibody
reagent is specific for a bridging antigen comprising a branched
structure.
105. The reagent composition of claim 97, wherein the antibody
reagent is specific for a bridging antigen comprising a peptide
comprising a non-natural residue.
106. The reagent composition of claim 105, wherein the non-natural
residue is a non-natural stereoisomer.
107. The reagent composition of claim 105, wherein the non-natural
residue is a .beta.-amino acid.
108. The reagent composition of claim 97, wherein the antibody
reagent is specific for a bridging antigen with a dissociation
constant of at most 1 nM.
109. The reagent composition of claim 92, wherein the antibody
reagent is specific for a cellular marker.
110. The reagent composition of claim 109, wherein the cellular
marker is selected from the group consisting of: 4-1BB, AFP, ALK1,
Amyloid A, Amyloid P, Androgen Receptor, Annexin A1, ASMA, BCA225,
BCL-1, BCL-2, BCL-6, BerEP4, Beta-Catenin, Beta-HCG, BG-8, BOB-1,
CA19-9, CA125, Calcitonin, Caldesmon, Calponin-1, Calretinin, CAM
5.2, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20,
CD21, CD22, CD23, CD25, CD30, CD31, CD33, CD34, CD38, CD42b, CD43,
CD45 LCA, CD45RO, CD47, CD56, CD57, CD61, CD68, CD79a, CD80, CD86,
CD99, CD117, CD138, CD163, CDX2, CEA, Chromogranin A, CMV, c-kit,
c-MET, c-MYC, Collagen Type IV, Complement 3c (C3c), COX-2, CXCR5,
CK1, CK5, CK6, CK7, CK8, CK14, CK18, CK17, CK19, CK20, CK903, CK
AE1, CK AE1/AE3, CSF-1, CSF-1R, D2-40, Desmin, DOG-1, E-Cadherin,
EGFR, EMA, ER, ERCC1, Factor VIII-RA, Factor XIIIa, Fascin, FoxP1,
FoxP3, Galectin-3, GATA-3, GATA-4, GCDFP-15, GCET1, GFAP, GITR,
Glycophorin A, Glypican 3, Granzyme B, HBME-1, Helicobacter pylori,
Hemoglobin A, Hep Par 1, HER2, HHV-8, HMB-45, HSV l/ll, ICOS,
IFNgamma, IgA, IgD, IgG, IgM, IL17, IL4, Inhibin, iNOS, Kappa Ig
Light Chain, Ki-67, LAG-3, Lambda Ig Light Chain, Lysozyme,
Mammaglobin A, MART-1/Melan A, Mast Cell Tryptase, MHC Class II,
MLH1, MOC-31, MPO, MSA, MSH2, MSH6, MUC1, MUC2, MUM1, MyoD1,
Myogenin, Myoglobin, Napsin A, Nestin, NSE, Oct-2, OX40, OX40L,
p16, p21, p27, p40, p53, p63, p504s, PAX-5, PAX-8, PD-1, PD-L1,
Perforin, PHH3, PIN-4, PLAP, PMS2, Pneumocystis jiroveci (carinii),
PR, PSA, PSAP, RCC, S-100, SMA, SMM, Smoothelin, SOX10, SOX11,
Surfactant Apoprotein A, Synaptophysin, TAG 72, T-bet, TdT,
Thrombomodulin, Thyroglobulin, TIA-1, TIM3, TRAcP, TTF-1,
Tyrosinase, Uroplakin, VEGF, VEGFR-2, Villin, Vimentin, and
WT-1.
111. A reagent compound comprising: a bridging oligonucleotide and
a latent crosslinker moiety.
112. The reagent compound of claim 111, wherein the bridging
oligonucleotide and the latent crosslinker moiety are linked by a
chemical coupling reaction through a conjugation moiety.
113. The reagent compound of claim 111, wherein the latent
crosslinker moiety comprises a phenol moiety.
114. The reagent compound of claim 113, wherein the latent
crosslinker moiety comprises a tyramine, a tyramide, or a
tyrosine.
115. A detectable oligonucleotide comprising: an oligonucleotide
complementary to a bridging oligonucleotide and a detectable
label.
116. A diagnostic kit comprising: a first reagent compound
comprising a bridging oligonucleotide and a latent crosslinker
moiety; a first detectable oligonucleotide comprising an
oligonucleotide complementary to the bridging oligonucleotide; and
instructions for use.
117. The kit of claim 116, wherein the first detectable
oligonucleotide comprises a detectable label.
118. The kit of claim 117, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
119. The kit of claim 118, wherein the detectable label is a
fluorophore.
120. The kit of claim 118, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
121. The kit of claim 116, further comprising a first
oligonucleotide reagent; wherein the first oligonucleotide reagent
comprises an oligonucleotide complementary to a target nucleic acid
and a crosslinker activation agent.
122. The kit of claim 121, wherein the crosslinker activation agent
comprises an enzyme.
123. The kit of claim 122, wherein the enzyme is a peroxidase, an
alkaline phosphatase, or a glucose oxidase.
124. The kit of claim 123, wherein the enzyme is a peroxidase.
125. The kit of claim 124, wherein the peroxidase is a horseradish
peroxidase or a soybean peroxidase.
126. The kit of claim 121, wherein the target nucleic acid is a
bridging oligonucleotide.
127. The kit of claim 126, wherein the target nucleic acid is the
bridging oligonucleotide of the first reagent compound.
128. The kit of claim 121, wherein the target nucleic acid is a
genetic marker.
129. The kit of claim 121, wherein the first oligonucleotide
reagent comprises added phenol moieties.
130. The kit of claim 129, wherein the added phenol moieties are
added tyrosine moieties.
131. The kit of claim 130, wherein the added tyrosine moieties are
residues in a peptide coupled to the first oligonucleotide
reagent.
132. The kit of claim 116, further comprising a second detectable
oligonucleotide and a second reagent compound.
133. The kit of claim 132, further comprising a first antibody
reagent and a second antibody reagent; wherein each antibody
reagent comprises an antibody and a crosslinker activation
agent.
134. A method for signal amplification comprising: providing a
first sample comprising a first target antigen; reacting the first
target antigen with a first antibody reagent, wherein the first
antibody reagent comprises an antibody specific for the first
target antigen and a crosslinker activation agent; reacting the
first antibody reagent with a first reagent compound, wherein the
first reagent compound comprises a bridging oligonucleotide and a
latent crosslinker moiety; and reacting the bridging
oligonucleotide with a first detectable oligonucleotide comprising
an oligonucleotide complementary to the bridging
oligonucleotide.
135. The method of claim 134, wherein the crosslinker activation
agent of the first antibody reagent comprises an enzyme.
136. The method of claim 135, wherein the enzyme is a peroxidase,
an alkaline phosphatase, or a glucose oxidase.
137. The method of claim 136, wherein the enzyme is a
peroxidase.
138. The method of claim 137, wherein the peroxidase is a
horseradish peroxidase or a soybean peroxidase.
139. The method of claim 134, wherein the first target antigen is a
cellular marker.
140. The method of claim 134, wherein the first target antigen is a
primary antibody specific for a cellular marker.
141. The method of claim 140, wherein the primary antibody is from
a different species than the antibody of the first antibody
reagent.
142. The method of claim 134, wherein the first target antigen is a
bridging antigen.
143. The method of claim 142, wherein the first antibody reagent is
specific for the bridging antigen with a dissociation constant of
at most 1 nM.
144. The method of claim 143, wherein the first detectable
oligonucleotide comprises a detectable label.
145. The method of claim 144, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
146. The method of claim 145, wherein the detectable label is a
fluorophore.
147. The method of claim 145, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
148. The method of claim 143, further comprising: detecting the
first detectable oligonucleotide.
149. A method for signal amplification comprising: providing a
first sample comprising a first target antigen; reacting the first
target antigen with a first antibody reagent, wherein the first
antibody reagent comprises an antibody specific for the first
target antigen and a crosslinker activation agent; reacting the
first antibody reagent with a first reagent compound, wherein the
first reagent compound comprises a bridging oligonucleotide and a
latent crosslinker moiety; reacting the bridging oligonucleotide of
the first reagent compound with a first oligonucleotide reagent,
wherein the first oligonucleotide reagent comprises an
oligonucleotide complementary to the bridging oligonucleotide of
the first reagent compound and a crosslinker activation agent; and
reacting the first oligonucleotide reagent with a second reagent
compound, wherein the second reagent compound comprises a bridging
oligonucleotide and a latent crosslinker moiety.
150. The method of claim 149, wherein the crosslinker activation
agent of the first oligonucleotide reagent comprises an enzyme.
151. The method of claim 150, wherein the enzyme is a peroxidase,
an alkaline phosphatase, or a glucose oxidase.
152. The method of claim 151, wherein the enzyme is a
peroxidase.
153. The method of claim 152, wherein the peroxidase is a
horseradish peroxidase or a soybean peroxidase.
154. The method of claim 149, wherein the first target antigen is a
cellular marker.
155. The method of claim 149, wherein the first target antigen is a
primary antibody specific for a cellular marker.
156. The method of claim 155, wherein the primary antibody is from
a different species than the antibody of the first antibody
reagent.
157. The method of claim 149, wherein the bridging oligonucleotide
of the first reagent compound and the bridging oligonucleotide of
the second reagent compound are the same.
158. The method of claim 149, further comprising: reacting the
bridging oligonucleotide of the second reagent compound with a
first detectable oligonucleotide complementary to the bridging
oligonucleotide.
159. The method of claim 158, wherein the first detectable
oligonucleotide comprises a detectable label.
160. The method of claim 159, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten.
161. The method of claim 160, wherein the detectable label is a
fluorophore.
162. The method of claim 160, wherein the detectable label is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
163. The method of claim 158, further comprising: detecting the
first detectable oligonucleotide.
164. A reagent composition comprising: an oligonucleotide reagent
comprising a crosslinker activation agent and an oligonucleotide
complementary to a target nucleic acid; and a reagent compound of
any one of claims 111-114.
165. The reagent composition of claim 164, wherein the crosslinker
activation agent comprises an enzyme.
166. The reagent composition of claim 165, wherein the enzyme is a
peroxidase, an alkaline phosphatase, or a glucose oxidase.
167. The reagent composition of claim 166, wherein the enzyme is a
peroxidase.
168. The reagent composition of claim 167, wherein the peroxidase
is a horseradish peroxidase or a soybean peroxidase.
169. The reagent composition of claim 164, wherein the target
nucleic acid is a bridging oligonucleotide.
170. The reagent composition of claim 169, wherein the target
nucleic acid is the bridging oligonucleotide of the reagent
compound.
171. The reagent composition of claim 164, wherein the target
nucleic acid is a genetic marker.
172. The reagent composition of claim 164, wherein the
oligonucleotide reagent comprises added phenol moieties.
173. The reagent composition of claim 172, wherein the added phenol
moieties are added tyrosine moieties.
174. The reagent composition of claim 173, wherein the added
tyrosine moieties are residues in a peptide coupled to the
oligonucleotide reagent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/363,821, filed on Jul. 18, 2016, the disclosure
of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The ability to detect low-expressing target markers, in some
cases at less than picogram levels, in cellular assays with high
sensitivity and specificity continues to be an unmet need. Such
approaches become even more important as the sample size of cells
and tissues available for analysis becomes smaller and smaller.
Furthermore, the ability to simultaneously detect multiple
low-expressing targets in a single assay would be of further
benefit.
[0003] The tyramide signal amplification (TSA) system is a
highly-sensitive analytical technique based on the ability of
horseradish peroxidase (HRP) to catalyze the deposition of large
amounts of tyramide proximal to an antigen-antibody complex. This
phenomenon was first observed in the late 1950s (Gross et al.
(1959) J. Biol. Chem. 234:1611) but only decades later was the
technique applied to the amplification of signals in immunoassays
(Bobrow et al. (1989) J. Immunol. Methods 125:279). The principle
of the reaction has since been adapted to immunohistochemistry
(IHC) and in situ hybridization (ISH) (Speel et al. (1997) J.
Histochem. Cytochem. 45:1439; Speel et al. (1999) Endocr. Pathol.
10:193; Raap et al. (1995) Hum. Mol. Genet. 4:529) to increase the
sensitivity of detection in these systems. While the TSA procedure
is used in IHC, it is especially pertinent in immunofluorescence
(IF) staining, since the HRP-catalyzed signal amplification yields
a linear increase in signal without altering the relative variation
in expression levels of the underlying target. The fluorescence
levels observed in an immunoassay thus correspond to the relative
levels of the original target antigen. TSA amplification in IHC
staining thus brings the signal to detectable levels, while TSA
amplification in IF staining not only boosts the signal, but also
reflects relative levels of target expression in the tissue.
[0004] Signal amplification with tyramide-modified fluorescent
substrates is 10-100 times more sensitive than a two-step labeling
protocol using a primary antibody and a fluorescently-labeled
anti-species secondary antibody. Sensitivity can be increased even
further using a three-step procedure with a biotinylated primary
antibody, a streptavidin-HRP conjugate, and a tyramide-modified
fluorophore. Variations in this technique using a hapten-labeled
reagent, e.g., a tyramide labeled with digoxigenin, dinitrophenyl,
or trinitrophenyl, and an HRP-modified antibody specific for the
respective hapten, have also been employed to detect targets in
tissues. See Speel et al. (1998) Histochem. Cell. Biol. 110:571.
The hapten-based methods produce signals greater than those
observed with HRP-labeled primary antibodies and tyramide-modified
fluorescent reagents but are less sensitive than the
biotin/streptavidin-based procedures. Tyramide-modified
fluorophores, tyramide-modified biotin, and tyramide-modified
haptens are available commercially from a variety of sources,
including ThermoFisher (Waltham, Mass.; www.thermofisher.com),
PerkinElmer (Waltham, Mass.; www.perkinelmer.com), and Biotium,
Inc. (Hayward, Calif.; www.biotium.com). U.S. Patent Application
Publication No. 2013/0109019A1 describes the use of
tyramide-modified haptens in signal amplification assays, including
immunohistochemical assays and in situ hybridizations, but no
optimization of the anti-hapten antibodies used in the assays was
reported.
[0005] While the three-step procedure described above with a
biotinylated primary antibody, a streptavidin-HRP conjugate, and a
tyramide-modified fluorophore provides extremely high sensitivity,
its use in immunohistochemistry is limited due to background
staining resulting from endogenous biotin in mammalian tissues.
Blocking strategies to minimize the background staining have been
employed with mixed success, and the use of this system has not
been widely adopted in any immunoassays.
[0006] Accordingly, despite the above approaches, there continues
to be a need for the development of improved reagent compounds,
compositions, methods, and kits that are more sensitive, more
specific, and more able to detect multiple antigens or nucleic
acids with high sensitivity and low background signal, ideally in a
single assay.
SUMMARY OF THE INVENTION
[0007] The present disclosure addresses these and other needs by
providing in one aspect reagent compounds that find utility in a
variety of bioanalytical assays. Specifically, according to this
aspect of the invention, the reagent compounds comprise a bridging
antigen or a bridging oligonucleotide and a latent crosslinker
moiety. In particular, in these reagent compound embodiments, the
bridging antigen is not a biotin, a hapten, or an antigenic
fluorophore.
[0008] In some embodiments, the bridging antigen comprises a
polymer. In more specific embodiments, the bridging antigen
comprises a peptide. In other embodiments, the bridging antigen
comprises a plurality of antigenic determinants, and more
specifically, each antigenic determinant in the plurality of
antigenic determinants is the same, or the plurality of antigenic
determinants comprises a linear repeating structure. Even more
specifically, the linear repeating structure may comprise a linear
repeating peptide structure. In some embodiments, the plurality of
antigenic determinants comprises at least three antigenic
determinants, and in some embodiments, the bridging antigen
comprises a branched structure.
[0009] In some embodiments, the bridging antigen comprises a
peptide comprising a non-natural residue, for example a non-natural
stereoisomer or a .beta.-amino acid. In some embodiments, the
bridging antigen and the latent crosslinker moiety are linked by a
chemical coupling reaction through a conjugation moiety.
[0010] In some embodiments, the latent crosslinker moiety comprises
a phenol moiety, more specifically the latent crosslinker moiety
comprises a tyramine, a tyramide, a tyrosine, or the like.
[0011] In another aspect, the present disclosure provides antibody
reagents comprising a crosslinker activation agent and an antibody
specific for a bridging antigen with high affinity. Alternatively,
the disclosure provides oligonucleotide reagents comprising a
crosslinker activation agent and an oligonucleotide complementary
to a bridging oligonucleotide. More specifically, the crosslinker
activation agent may comprise an enzyme, such as a peroxidase, an
alkaline phosphatase, or a glucose oxidase, for example a
horseradish peroxidase or a soybean peroxidase.
[0012] In some embodiments, the antibody of the instant antibody
reagent is specific for any of the above-described bridging
antigens, including hapten bridging antigens, with high
affinity.
[0013] In some embodiments, the antibody of the instant antibody
reagents is specific for the bridging antigen with a dissociation
constant of at most 1 nM.
[0014] In some embodiments, the crosslinker activation agent and
the antibody or the oligonucleotide are linked by a chemical
coupling reaction through a conjugation moiety.
[0015] In some embodiments, the antibody reagents or
oligonucleotide reagents comprise added phenol moieties, for
example, added tyrosine moieties, including added tyrosine moieties
that may be residues in a peptide coupled to the antibody reagent
or oligonucleotide reagent.
[0016] In yet another aspect, the present disclosure provides
detectable antibodies comprising an antibody specific for a
bridging antigen, including any of the above-described bridging
antigens, with high affinity, and a detectable label.
Alternatively, the disclosure provides detectable oligonucleotides
comprising an oligonucleotide complementary to a bridging
oligonucleotide, including any of the above-described bridging
oligonucleotides, and a detectable label.
[0017] In still other aspects, the disclosure provides diagnostic
kits comprising a reagent compound comprising a bridging antigen or
bridging oligonucleotide and a latent crosslinker moiety, a
detectable antibody or a detectable oligonucleotide, and
instructions for use. In some embodiments, the diagnostic kit
further comprises an antibody reagent or an oligonucleotide
reagent, wherein the antibody reagent comprises an antibody and a
crosslinker activation agent and the oligonucleotide reagent
comprises an oligonucleotide and a crosslinker activation agent. In
specific embodiments, the antibody reagent is specific for a
bridging antigen with high affinity. In other specific embodiments,
the antibody reagent is specific for a cellular marker. In still
other specific embodiments, the antibody reagent is specific for a
cross-species immunoglobulin.
[0018] In some embodiments, the detectable antibody or detectable
oligonucleotide of the instant kits comprises a detectable label
such as, for example, a fluorophore, an enzyme, an upconverting
nanoparticle, a quantum dot, or a detectable hapten.
[0019] According to yet another aspect, the disclosure provides
methods for signal amplification comprising providing a first
sample comprising a first target antigen, reacting the first target
antigen with a first antibody reagent, wherein the first antibody
reagent comprises an antibody specific for the first target antigen
and a crosslinker activation agent, reacting the first antibody
reagent with a first reagent compound, wherein the first reagent
compound comprises a bridging antigen or a bridging oligonucleotide
and a latent crosslinker moiety, and reacting the bridging antigen
or bridging oligonucleotide with a first detectable antibody
comprising an antibody specific for the bridging antigen with high
affinity or a first detectable oligonucleotide comprising an
oligonucleotide complementary to the bridging oligonucleotide.
[0020] In some embodiments, the methods further comprise detecting
the first detectable antibody or the first detectable
oligonucleotide.
[0021] In yet another aspect, the disclosure provides reagent
compositions comprising a reagent compound and an antibody reagent
comprising a crosslinker activation agent and an antibody or an
oligonucleotide reagent comprising a crosslinker activation agent
and an oligonucleotide, wherein the reagent compounds are as
described above, and wherein the antibody reagent and the
oligonucleotide reagent are as also described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A: Schematic representation of an exemplary method for
one-round signal amplification using an unmodified primary
antibody, an HRP-labeled cross-species secondary antibody, and a
tyramide-labeled bridging antigen reagent compound. The amplified
bridging antigen is labeled with a detectable antibody specific for
the bridging antigen, as shown in step D.
[0023] FIG. 1B: Schematic representation of an alternative
exemplary method for one-round signal amplification using an
unmodified primary antibody, an HRP-labeled cross-species secondary
antibody, and a tyramide-labeled bridging oligonucleotide reagent
compound. In this method, the amplified bridging oligonucleotide is
labeled with a detectable oligonucleotide complementary to the
bridging oligonucleotide, as shown in step D.
[0024] FIG. 2A: Schematic representation of a variant exemplary
method for one-round signal amplification using a bridging
antigen-labeled primary antibody, an HRP-labeled anti-bridging
antigen secondary antibody, and a tyramide-labeled bridging antigen
reagent compound. The bridging antigen of the tryamide-labeled
reagent compound may be the same as or different from the bridging
antigen associated with the bridging antigen-labeled primary
antibody. As in FIG. 1A, the amplified bridging antigen is labeled
with a detectable antibody specific for the bridging antigen, as
shown in step D.
[0025] FIG. 2B: Schematic representation of another variant
exemplary method for one-round signal amplification using a
bridging antigen-labeled primary antibody, an HRP-labeled
anti-bridging antigen secondary antibody, and a tyramide-labeled
bridging oligonucleotide reagent compound. As in FIG. 1B, in this
method the amplified bridging oligonucleotide is labeled with a
detectable oligonucleotide complementary to the bridging
oligonucleotide, as shown in step D.
[0026] FIG. 2C: Schematic representation of a multiplexed variant
of the one-round signal amplification method shown in FIG. 2A,
where a second target antigen is reacted with a second bridging
antigen-labeled primary antibody in step D. The second bridging
antigen is reacted with a second HRP-labeled anti-bridging antigen
secondary antibody (step E), and this complex is reacted with a
second tyramide-labeled bridging antigen reagent compound (step F).
The bridging antigens of the first and second tyramide-labeled
reagent compounds are designated as triangles and circles,
respectively. Not shown in this scheme is the reaction of these
bridging antigens with detectable antibodies specific for those
bridging antigens.
[0027] FIG. 3A: Schematic representation of the second round in a
two-round signal amplification method using a tyramide-labeled
bridging antigen. In this example, steps A through C are the same
as those shown in FIG. 1A, FIG. 2A, and
[0028] FIG. 2C, but the amplification step is repeated using a
second treatment round with an antibody reagent specific for the
bridging antigen and a tyramide-labeled bridging antigen, as shown
in steps D and E. As in FIGS. 1A and 2A, the amplified bridging
antigen is labeled with a detectable antibody specific for the
bridging antigen, as shown in step F.
[0029] FIG. 3B: Schematic representation of a two-round signal
amplification method using a tyramide-labeled bridging
oligonucleotide and an oligonucleotide reagent. In this example,
steps A through C are the same as those shown in either FIG. 1B or
FIG. 2B, where an antibody reagent is bound to a target antigen
(step B), and multiple copies of a bridging oligonucleotide are
immobilized by crosslinking to the sample surface in the vicinity
of the target antigen (step C). In step D, however, the bridging
oligonucleotide is reacted with an HRP-labeled oligonucleotide
reagent complementary to the bridging oligonucleotide. Another
reaction round with the reagent compound (step E) further amplifies
the number of bridging oligonucleotides immobilized on the surface.
Reaction of the amplified bridging oligonucleotides with a
complementary detectable oligonucleotide for subsequent detection
is not shown in this drawing.
[0030] FIG. 3C: Schematic representation of a two-round signal
amplification method using a tyramide-labeled bridging
oligonucleotide and a single-stranded rolling circle template
complementary to the bridging oligonucleotide. In this example,
steps A through C are the same as those shown in either FIG. 1B or
FIG. 2B, where an antibody reagent is bound to a target antigen
(step B), and multiple copies of a bridging oligonucleotide are
immobilized by crosslinking to the sample surface in the vicinity
of the target antigen (step C). In step D, however, the bridging
oligonucleotide is reacted with a single-stranded circular template
that includes a sequence complementary to the bridging
oligonucleotide. Addition of a DNA polymerase and the 4 dNTPs
results in extension of the bridging oligonucleotide by rolling
circle amplification (step E), generating further binding sites for
a complementary detectable oligonucleotide (step F).
[0031] FIG. 4: Schematic representation of an in situ hybridization
method for identifying a target nucleic acid using multiple short
oligonucleotide probes coupled to a single bridging antigen or
bridging oligonucleotide (represented by triangles). Only a single
amplification step is shown in this scheme. Reaction of the
amplified bridging oligonucleotides with a complementary detectable
oligonucleotide for subsequent detection is not shown in this
drawing
[0032] FIG. 5: Schematic representation of an in situ hybridization
method for identifying a target nucleic acid using multiple
oligonucleotide probes coupled to different bridging antigens or
bridging oligonucleotides (represented by triangles and circles).
Reaction of the different amplified bridging antigens or
oligonucleotides with complementary detectable antibodies or
oligonucleotides is not shown in this drawing.
[0033] FIG. 6: Schematic representation of the use of an antibody
reagent comprising added phenol moieties.
[0034] FIG. 7: Exemplary synthetic route used to prepare N-terminal
linked tyramide-modified reagent compounds using a high-efficiency
conjugation moiety.
[0035] FIG. 8: Alternative synthetic route to prepare N-terminal
linked tyramide-modified reagent compounds via solid phase peptide
synthesis.
[0036] FIGS. 9A-9E: Exemplary staining of a triple-positive breast
cancer tissue sample using a mouse primary antibody specific for
Ki-67 and various tyramide-modified reagent compounds and various
amplification steps.
[0037] FIGS. 10A-10B: Exemplary one-round and two-round staining of
Ki-67 on triple-positive breast cancer tissue using an anti-Ki-67
antibody labeled with the PEP6 bridging antigen at 10 pg/.mu.L.
[0038] FIGS. 11A-11D: Staining of Ki-67 on triple-positive breast
cancer tissue with one-round and two-round amplification methods
using tyramide-biotin and a tyramide-peptide bridging antigen with
anti-Ki-67-HRP at 100 pg/.mu.L.
[0039] FIGS. 12A-12D: Staining of Ki-67 on triple-positive breast
cancer tissue with one-round and two-round amplification methods
using tyramide-biotin and a tyramide-peptide bridging antigen with
anti-Ki-67-HRP at 10 pg/.mu.L.
[0040] FIGS. 13A-13H: Staining of HER2 on triple-positive breast
cancer tissues comparing one round vs. two rounds of
anti-HER2-PEP5/anti-PEP5-HRP/tyramide-PEP5/anti-PEP5-Dy650 at
decreasing concentrations of anti-HER2 antibodies.
[0041] FIGS. 14A-14C: Comparison of the staining intensity of a
primary rabbit antibody targeting the estrogen receptor (ER) on
triple-positive breast cancer tissue. The HRP-labeled anti-rabbit
secondary antibodies used for the amplification step contained
increasing amounts of a poly-tyrosine peptide to increase
staining.
[0042] FIGS. 15A-15B: Staining of HER2 on triple-positive breast
cancer tissue with a rabbit anti-HER2 primary antibody, an
anti-rabbit-HRP secondary antibody, and tyramide-labeled PEP5 and
PEP5-3.times. bridging antigens.
DETAILED DESCRIPTION OF THE INVENTION
Reagent Compounds
[0043] The instant disclosure provides in one aspect
high-performance reagent compounds comprising a bridging antigen or
bridging oligonucleotide and a latent crosslinker moiety. Such
compounds find utility in reagent compositions, methods for signal
amplification, and diagnostic kits, where they enable the
amplification of detectable signals by the targeted immobilization
of multiple copies of a bridging antigen or bridging
oligonucleotide in the vicinity of a cellular marker, either on or
in a sample of interest. In particular, the reagent compounds find
utility in combination with antibody reagents and oligonucleotide
reagents, to be described in detail below, that comprise an
antibody or an oligonucleotide and a crosslinker activation agent.
The antibody or oligonucleotide component of the reagent
specifically associates the crosslinker activation agent at a
specific location on or in a sample of interest, where the
crosslinker activation agent catalytically activates the latent
crosslinker functionality of the reagent compound, thereby causing
immobilization of multiple--ideally many multiple--bridging
antigens or bridging oligonucleotides to the sample surface in the
vicinity of the crosslinker activation agent.
[0044] In specific embodiments, the latent crosslinker moiety of
the instant reagent compounds comprises a phenol moiety, and more
specifically, the latent crosslinker moiety comprises a tyramine, a
tyramide, a tyrosine, or the like. Tyramide signal amplification
(TSA) is a well-known and powerful method for the amplification of
signals in biological assays such as immunohistochemical assays,
immunofluorescence assays, and in situ hybridizations. According to
this technique, horseradish peroxidase (HRP), or another suitable
oxidase enzyme, is attached to an antibody or other targeting agent
capable of binding the oxidase enzyme to a location of interest.
The oxidase, typically in combination with added hydrogen peroxide,
reacts catalytically with tyramide-modified compounds in the assay
solution to produce short-lived free radicals that can react with
tyrosine moieties, or other reactive groups, on proteins or other
reactive molecules proximal to the bound oxidase. By the covalent
attachment of a binding agent, such as a biotin moiety, to the
tyramide compound, amplification of binding sites, such as biotin
binding sites, to the sample is achieved. The amplification of
biotin binding sites on the sample increases the binding of
detectable labels, such as fluorescent streptavidin, to the sample,
and thereby increases the signal.
[0045] The oxidation of tyrosine by HRP has more generally been
exploited previously both to conjugate and to immobilize proteins.
For example, Minamihata et al. (2011) Bioconjugate Chem., 22, 2332,
have described the conjugation of proteins through HRP-mediated
catalysis of proteins genetically engineered to incorporate
tyrosine moieties. Furthermore, Endrizzi et al. (2006) Langmuir 22,
11305, have described the immobilization of green fluorescent
protein ("GFP") genetically engineered to incorporate a
tyrosine-His6 tag to a tyrosine-methacylate-based microbead. These
examples support the general principle of enzyme-catalyzed
crosslinking of attached moieties to reactive surfaces and the
utility of such approaches in amplified bioassays.
[0046] Tyramide signal amplification and HRP-catalyzed protein
conjugation are two specific examples of a more general approach
that has been termed "catalyzed reporter deposition" or "CARD".
See, e.g., Bobrow et al. (1989) J. Immunol. Methods
125(1-2):279-85. This method involves the use of a so-called
"analyte-dependent reporter enzyme" (ADRE) to catalyze the
deposition of a reporter reagent on the surface in a solid-phase
immunoassay. According to the original approach, also referred to
as an "analyte dependent enzyme activation system" (ADEAS), the
reporter reagent or "conjugate" is chosen based on its ability to
be activated by a particular enzyme. See, e.g., U.S. Pat. Nos.
5,196,306; 5,583,001; 5,688,966; 5,731,158; 5,767,287; 5,863,748;
6,372,937; and 6,593,100. When HRP is used as the enzyme in such
systems, conjugates containing a phenolic moiety can be activated
by the HRP to generate an activated phenolic substrate. Without
intending to be bound by theory, it is believed that the activated
phenolic substrate reacts with electron-rich residues, such as the
side chains of tyrosines and tryptophans on proteins associated
with the assay surface, to form covalent adducts. As noted in U.S.
Pat. No. 5,196,306, other enzyme/conjugate pairs can be used in
these methods, including conjugates that result in crosslinks with
both endogenous surface targets (i.e., proteins associated with the
assay surface) and exogenous targets that are applied to the assay
surface prior to the enzyme activation.
[0047] As would be understood from the above description, the
latent crosslinker moiety of the instant reagent compounds is
chosen in combination with the choice of a crosslinker activation
agent in the antibody or oligonucleotide reagent with which it is
used. Suitable latent crosslinker moieties are chemical moieties
that upon activation will react with targets on the surface of an
assay, such that the resulting crosslinks will be sufficiently
stable to remain attached during the subsequent detection steps of
the assay. In addition, activation of the latent crosslinker moiety
should occur sufficiently rapidly that the assays can be completed
on a reasonable time scale, and the activated crosslinker moiety
should be sufficiently reactive to couple readily with suitable
targets in the vicinity of the crosslinker activation agent.
Moreover, the activated crosslinker moiety should be spontaneously
deactivated faster than the rate that the reagent compound diffuses
away from the crosslinker activation agent, so that the activated
reagent compound does not crosslink reactive targets that are not
in the vicinity of the crosslinker activation agent.
[0048] In addition to a latent crosslinker moiety, the reagent
compounds of the instant disclosure also comprise a bridging
antigen or a bridging oligonucleotide. Bridging antigens are chosen
to be recognizable by an antibody, typically an antibody reagent or
a detectable antibody, and ideally at high affinity, as will be
further described below. The structure of the bridging antigen is
therefore limited only by molecules that are capable of eliciting
an immune response in a suitable animal or that can be used to
generate suitable antibodies by another means. Bridging
oligonucleotides are chosen to be recognizable by a complementary
oligonucleotide, typically an oligonucleotide reagent or a
detectable oligonucleotide, and ideally at high affinity, through
specific base pairing interactions, as is well understood in the
art. The length, base sequence, chemical backbone, and other
structural features of each member of a particular oligonucleotide
pair, as well as the specific hybridization conditions (e.g., pH,
buffer salts, temperature, etc.) used to associate the two
components of the pair, are chosen to modulate the strength of this
association, as is also well understood in the art.
[0049] In some embodiments, the bridging antigen of the instant
disclosure is or comprises a synthetic bridging antigen. In some
embodiments, the bridging antigen is or comprises a natural
product. In some embodiments, the bridging antigen is or comprises
a polymer, including a non-repeating polymer, a biological polymer
(e.g., a polypeptide, a nucleic acid, a carbohydrate, or the like),
a non-biological polymer, a multimerized small molecule, a
biological non-polymer (e.g., a lipid), or any other suitable
molecule, so long as the molecule is capable of eliciting an immune
response and being bound by a suitable antibody, either alone or in
combination with a carrier protein, such as keyhole limpet
hemocyanin, or any other suitable vehicle. In specific embodiments,
the bridging antigen is or comprises a peptide. In some
embodiments, the bridging antigen is or comprises a biotin, a
non-peptidic, small-molecule antigen (also known as a hapten), or
an antigenic fluorophore. In other embodiments, however, the
bridging antigen is not a biotin, a hapten, or an antigenic
fluorophore. In some embodiments, the bridging antigen is not a
molecule that occurs naturally in a normal cell. As would be
understood by those of ordinary skill in the art, these embodiments
are of particular advantage in minimizing background signal from
the binding of antibody reagents to naturally-occurring molecules
on a sample surface and the resultant amplification of background
signal due to such background binding.
[0050] Peptides, either synthetic or isolated from natural sources,
have been used extensively to generate specific, high-affinity
antibodies by various means, as is widely known and understood by
those of ordinary skill in the art. The instant inventors have
usefully discovered that peptidic bridging antigens and monoclonal
antibodies specific for such antigens at high affinity are
particularly useful for providing high sensitivity and low
backgrounds in the amplified assays described herein. Other
antigenic molecules, including all of the bridging antigens
described above, and antibodies specific for those antigenic
molecules at high affinity, including all of the antibodies
described above, are likewise useful in the instant amplified
assays.
[0051] The range of structural variation possible with peptidic
antigens is nearly limitless, thus making them ideally suited for
use as bridging antigens in the instant reagent compounds.
Furthermore, synthetic peptides can be designed to include reactive
groups to facilitate their coupling to antibodies or other chemical
entities, for example by including amino acid residues or other
linking moieties incorporated on the C- or N-termini or internally
during solid phase peptide synthesis or post-synthetically with
desirable reactive properties within the peptide sequence. Peptidic
bridging antigens may be of any size and may contain any suitable
amino acid or other residue, both natural and artificial. They may
be linear, circular, or branched. The peptidic bridging antigens
are limited in these embodiments only by their ability to be
conjugated to a latent crosslinker moiety or antibody and to be
recognizable by a suitable antibody reagent or detectable
antibody.
[0052] In some embodiments, the bridging antigen is a peptide
comprising a non-natural residue. For example, the bridging antigen
may comprise a non-natural stereoisomer, such as a D-amino acid. In
some embodiments, the non-natural residue may be a non-natural
amino acid, such as a .beta.-amino acid or the like. In some
embodiments, the residues of the bridging antigen may be coupled
using non-peptidic bonding, as would be understood by those of
ordinary skill in the art. Novel small-molecule antigens, also
known as haptens, and conjugates of the haptens, as well as
antibodies against the haptens, and methods of using these
reagents, for example in immunohistochemical and in situ
hybridization techniques, are disclosed in U.S. Pat. Nos.
7,695,929; 8,618,265; 8,846,320; and 9,103,822. These, and other,
haptens can accordingly be adapted for use as bridging antigens in
reagent compounds by coupling them to a latent crosslinker moiety.
See also U.S. Patent Application Publication No. 2013/0109019A1. It
is particularly important when using a hapten as a bridging antigen
in the instant reagent compounds, compositions, kits, and methods,
however, that the corresponding antibody be a high affinity
antibody or that it be optimized to become a high affinity
antibody. See below for a description of antibody optimization.
[0053] In order to increase the number of antibody binding sites
per reagent compound, it may be advantageous in some cases for a
single bridging antigen to comprise a plurality of antigenic
determinants or epitopes. Multiplicity of antigenic determinants in
a bridging antigen may increase the number of antibody reagents or
detectable antibodies able to bind to the bridging antigen and thus
the sensitivity of assays using the reagent compound. In some
embodiments, the plurality of antigenic determinants may comprise
multiple copies of the same antigenic determinant, whereas in some
embodiments, the plurality of antigenic determinants may comprise
different antigenic determinants. In some embodiments, the
plurality of antigenic determinants may comprise a linear repeating
structure. More specifically, the linear repeating structure may be
a linear repeating peptide structure. In some embodiments, the
plurality of antigenic determinants may comprise at least two
antigenic determinants, at least three antigenic determinants, at
least four antigenic determinants, at least six antigenic
determinants, or even more antigenic determinants.
[0054] In some embodiments, the bridging antigen may comprise a
branched structure. For example, the branched structure may
comprise a dendrimeric structure or the like, such as, for example,
other polymerized constructs, as would be understood by those of
ordinary skill in the art.
[0055] Furthermore, it should be understood that a bridging antigen
comprising a plurality of antigenic determinants may comprise one
or more polyethylene glycol linkers, or the like, between the
antigenic determinants, for example between peptide antigenic
determinants.
[0056] In some embodiments, the peptide antigenic determinants
comprise at least four, at least six, at least eight, at least ten,
at least 15, at least 20, or even more amino acid residues per
antigenic determinant.
[0057] Exemplary bridging antigens are described in U.S. patent
application Ser. No. 15/017,626 and PCT International Application
No. PCT/US2016/016913, both of which were filed on Feb. 6, 2016,
and both of which are incorporated herein by reference in their
entireties.
[0058] As noted above, the bridging oligonucleotides of the instant
reagent compounds, compositions, kits, and methods are chosen to be
complementary to the oligonucleotide reagent and/or detectable
oligonucleotide with which they are paired. In some embodiments,
the bridging oligonucleotide is a synthetic oligonucleotide. In
some embodiments, the bridging oligonucleotide is a locked nucleic
acid (LNA), a peptide nucleic acid (PNA), or the like.
[0059] The bridging antigen or bridging oligonucleotide and the
latent crosslinker moiety are typically attached to one another by
a chemical linkage. Attachment of the two components to one another
can occur as part of the process of synthesizing one or the other
of the components, or the two components can be attached to one
another by chemical coupling after they have been separately
synthesized. In the case of a synthetic peptidic bridging antigen
or a synthetic bridging oligonucleotide, the latent crosslinker
moiety can be attached to the peptide or oligonucleotide either
during or after a solid-state peptide or oligonucleotide synthesis
reaction. It should be understood that the coupling of a bridging
antigen or oligonucleotide to a latent crosslinker moiety should
not significantly affect the ability of the bridging antigen or
oligonucleotide to be recognized by their binding partners, nor
should the coupling significantly affect the ability of the latent
crosslinker moiety to be activated by a crosslinker activation
agent. It is also desirable that neither the bridging antigen, the
bridging oligonucleotide, nor the latent crosslinker moiety
themselves have interfering absorbance or fluorescence, so as to
avoid any interfering signals. Furthermore, bridging antigens,
bridging oligonucleotides, and latent crosslinker moieties should
preferably be available at high purity and ideally at low cost.
[0060] Where the bridging antigen or oligonucleotide and the latent
crosslinker moiety are prepared from separate molecular entities,
it should be understood that the coupling of the bridging antigen
or oligonucleotide and the latent crosslinker moiety may be
achieved in a wide variety of ways, depending on the desired
outcome. If control of the location and degree of coupling of the
bridging antigen or oligonucleotide to the latent crosslinker
moiety is not important, non-specific chemical crosslinkers may be
used to achieve the coupling. It is generally desirable, however,
for the bridging antigen or oligonucleotide to be coupled to the
latent crosslinker moiety in a controlled and specific manner, and
the choice of coupling method and agent can affect the location,
degree, and efficiency of the coupling.
[0061] In some reagent compound embodiments, the bridging antigen
or oligonucleotide and the latent crosslinker moiety are coupled by
a chemical coupling reaction through a conjugation moiety. In
specific embodiments, the bridging antigen or oligonucleotide and
the latent crosslinker moiety are coupled by a high-efficiency
conjugation moiety. Because the reagent compounds may be
synthesized with relatively low molar concentrations of starting
materials, and because those starting materials may be expensive
and available in relatively small chemical quantities, it is highly
desirable that formation of the conjugation moiety be as efficient
and specific as possible and that its formation be complete, or
nearly complete, at low molar concentrations of reactants.
Specifically, it is desirable that the conjugation moiety be
capable of coupling a bridging antigen or oligonucleotide and a
latent crosslinker moiety with rapid kinetics and/or high
association constants and that the association reaction therefore
be as efficient as possible in terms of its completion.
[0062] The high-efficiency conjugation moieties of the instant
reagent compounds are typically formed, as described in more detail
below, by separate modification of each component of the reagent
compound with complementary conjugating reagents. The complementary
conjugating reagents additionally include a further reactive
moiety, for example a thiol-reactive or an amino-reactive moiety,
that allows the conjugating reagents to be attached to the relevant
reagent component, for example to the bridging antigen or
oligonucleotide and to the latent crosslinker moiety. After the
bridging antigen or oligonucleotide and the latent crosslinker
moiety have been modified by the respective complementary
conjugating reagents, the complementary conjugating features on the
modified components associate with one another in a highly
efficient and specific manner to form the conjugation moiety.
[0063] Depending on the situation, the high-efficiency conjugation
moiety of the instant reagent compounds may be a covalent or
non-covalent conjugation moiety. In specific embodiments, the
high-efficiency conjugation moiety is a covalent conjugation
moiety, for example, a hydrazone, an oxime, or another suitable
Schiff base moiety. Non-limiting examples of such conjugation
moieties may be found, for example, in U.S. Pat. No. 7,102,024,
which is incorporated by reference herein in its entirety for all
purposes. These conjugation moieties may be formed by reaction of a
primary amino group on the conjugating reagent attached to one
component of the reagent (e.g., a latent crosslinker moiety) with a
complementary carbonyl group on the conjugating reagent attached to
the other component of the reagent (e.g., a bridging antigen or
oligonucleotide).
[0064] For example, hydrazone conjugation moieties may be formed by
the reaction of a hydrazino group, or a protected hydrazino group,
with a carbonyl moiety. Exemplary hydrazino groups include
aliphatic, aromatic, or heteroaromatic hydrazine, semicarbazide,
carbazide, hydrazide, thiosemicarbazide, thiocarbazide, carbonic
acid dihydrazine, or hydrazine carboxylate groups. See, for
example, U.S. Pat. No. 7,102,024. Oxime conjugation moieties may be
formed by the reaction of an oxyamino group, or a protected
oxyamino group, with a carbonyl moiety. Exemplary oxyamino groups
are described below. The hydrazino and oxyamino groups may be
protected by formation of a salt of the hydrazino or oxyamino
group, including but not limited to, mineral acid salts, such as
but not limited to hydrochlorides and sulfates, and salts of
organic acids, such as but not limited to acetates, lactates,
malates, tartrates, citrates, ascorbates, succinates, butyrates,
valerates and fumarates, or any amino or hydrazino protecting group
known to those of skill in the art (see, e.g., Greene et al. (1999)
Protective Groups in Organic Synthesis (3rd Ed.) (J. Wiley Sons,
Inc.)). The carbonyl moiety used to generate a Schiff base
conjugation moiety is any carbonyl-containing group capable of
forming a hydrazone or oxime linkage with one or more of the above
hydrazino or oxyamino moieties. Preferred carbonyl moieties include
aldehydes and ketones, in particular aromatic aldehydes and
ketones. In particularly preferred embodiments of the instant
disclosure, the high-efficiency conjugation moiety is formed by the
reaction of an oxyamino-containing component and an aromatic
aldehyde-containing component in the presence of aniline catalysis.
See Dirksen et al. (2006) Angew. Chem. 45:7581-7584 (DOI:
10.1002/anie.200602877).
[0065] The high-efficiency conjugation moiety of the instant
reagent compounds may alternatively be formed by a "click"
reaction, for example the copper-catalyzed reaction of an
azide-substituted component with an alkyne-substituted component to
form a triazole conjugation moiety. See Kolb et al. (2001) Angew.
Chem. Int. Ed. Engl. 40:2004; Evans (2007) Aus. J. Chem. 60:384.
Copper-free variants of this reaction, for example the
strain-promoted azide-alkyne click reaction, may also be used to
form the high-efficiency conjugation moiety. See, e.g., Baskin et
al. (2007) Proc. Natl Acad. Sci. U.S.A. 104:16793-97. Other click
reaction variants include the reaction of a tetrazine-substituted
component with either an isonitrile-substituted component
(Stockmann et al. (2011) Org. Biomol. Chem. 9:7303) or a strained
alkene-substituted component (Karver et al. (2011) Bioconjugate
Chem. 22:2263).
[0066] The basic features of a click reaction are well understood
by those of ordinary skill in the art. See Kolb et al. (2001)
Angew. Chem. Int. Ed. Engl. 40:2004. Useful click reactions include
generally but are not limited to [3+2] cycloadditions, such as the
Huisgen 1,3-dipolar cycloaddition, and in particular the
Cu(I)-catalyzed stepwise variant, thiol-ene click reactions,
Diels-Alder reactions and inverse electron demand Diels-Alder
reactions, [4+1] cycloadditions between isonitriles (isocyanides)
and tetrazines, nucleophilic substitutions, especially to small
strained rings like epoxy and aziridine compounds,
carbonyl-chemistry-like formation of ureas, and some addition
reactions to carbon-carbon double bonds. Any of the above reactions
may be used without limitation to generate a covalent
high-efficiency conjugation moiety in the instant reagent
compounds.
[0067] In some embodiments, the conjugation moiety of the instant
reagent compounds comprises a cleavable linker. Exemplary cleavable
linkers usefully included in the instant high-efficiency
conjugation moiety are known in the art. See, e.g., Leriche et al.
(2012) Bioorg. Med. Chem. 20:571-582
(doi:10.1016/j.bmc.2011.07.048). Inclusion of a cleavable linker in
the high-efficiency conjugation moiety allows for the selective
cleavage of the bridging antigen or oligonucleotide from the latent
crosslinker moiety in the instant reagent compounds. Such selective
cleavage may be advantageous in some assay methods, for example
where release of a bridging antigen or oligonucleotide from the
associated crosslinker moiety is desired.
[0068] In other embodiments, the high-efficiency conjugation moiety
is a non-covalent conjugation moiety. Non-limiting examples of a
non-covalent conjugation moiety include an oligonucleotide
hybridization pair or a protein-ligand binding pair. In specific
embodiments, the protein-ligand binding pair is an avidin-biotin
pair, a streptavidin-biotin pair, or another protein-biotin binding
pair (see generally Avidin-Biotin Technology, Meth. Enzymol. (1990)
volume 184, Academic Press; Avidin-Biotin Interactions: Methods and
Applications (2008) McMahon, ed., Humana; Molecular Probes.RTM.
Handbook, Chapter 4 (2010)), an antibody-hapten binding pair (see
generally Molecular Probes.RTM. Handbook, Chapter 4 (2010)), an
S-peptide tag-S-protein binding pair (Kim and Raines (1993) Protein
Sci. 2:348-56), or any other high-affinity peptide-peptide or
peptide-protein binding pair. Such high-affinity non-covalent
conjugation moieties are well known in the art. Reactive versions
of the respective conjugating pairs, for example thiol-reactive or
amino-reactive versions, are also well known in the art. These
conjugating reagents may be used to modify the respective bridging
antigen or oligonucleotide and latent crosslinker moiety. The
modified bridging antigen or oligonucleotide and latent crosslinker
moiety may then be mixed in order to allow the complementary
features, for example the oligonucleotide hybridization pair or the
protein-ligand binding pair, to associate with one another and form
a non-covalent high-efficiency conjugation moiety. All of the
above-described covalent and non-covalent linking groups are
capable of highly efficient association reactions and are thus well
suited for use in generation of the instant reagent compounds.
[0069] In some embodiments, the high-efficiency conjugation moiety
is at least 50%, 80%, 90%, 93%, 95%, 97%, 98%, 99%, or even more
efficient in coupling the bridging antigen or oligonucleotide and
the latent crosslinker moiety. In more specific embodiments, the
high-efficiency conjugation moiety is at least 50%, 80%, 90%, 93%,
95%, 97%, 98%, 99%, or even more efficient at a reagent
concentration of no more than 0.5 mg/mL. In some embodiments, the
efficiencies are achieved at no more than 0.5 mg/mL, no more than
0.2 mg/mL, no more than 0.1 mg/mL, no more than 0.05 mg/mL, no more
than 0.02 mg/mL, no more than 0.01 mg/mL, or even lower reagent
concentrations.
Crosslinker Activation Agents
[0070] It should be understood that the crosslinker activation
agents of the instant antibody and oligonucleotide reagents can be
any agent capable of activating the latent crosslinker moiety of a
suitable reagent compound in a catalytic manner. Suitable
crosslinker activation agent/latent crosslinker moiety combinations
include without limitation the combinations shown in Table 1 below.
Also shown in this table is the surface target of each of the
activated crosslinker moieties.
TABLE-US-00001 TABLE 1 Exemplary Reagent Combinations Crosslinker
activation agent Latent crosslinker moiety Surface target HRP
Substituted phenols Endogenous proteins or blocking proteins HRP
3-methyl-2-benzothiazolinone 3-(dimethyl-amino)benzoic acid (DMAB)
hydrazone (MBTH) .beta.-Galactosidase
.beta.-Galactopyranosyl-glycoside Antibody to deglycosylated moiety
Alkaline phosphatase NADP NAD binding proteins Alkaline phosphatase
Substituted phosphate compounds Antibody to dephosphorylated
compounds Alkaline phosphatase Phosphorylated biotin
Avidin/streptavidin
[0071] Further examples of reagent combinations suitable for use in
the instant signal amplification methods, including crosslinker
activation agents involving multiple-enzyme combinations, are
provided in U.S. Pat. Nos. 5,196,306; 5,583,001; 5,688,966;
5,731,158; 5,767,287; 5,863,748; 6,372,937; and 6,593,100. It
should be understood that the crosslinker activation agents of the
instant disclosure should be construed broadly to include any
agent, not just an enzyme, that is capable of activating the latent
crosslinker moiety of a reagent compound in a catalytic manner with
suitable reaction properties. In specific embodiments, however, the
crosslinker activation agent of the instant antibody and
oligonucleotide reagents comprises an enzyme. More specifically,
the enzyme may be a peroxidase, an alkaline phosphatase, or a
glucose oxidase. Even more specifically, the enzyme may be a
peroxidase, such as HRP.
Antibody Reagents
[0072] As noted above, the antibody reagents of the instant
disclosure generally comprise an antibody and a crosslinker
activation agent, where the antibody component serves to associate
the crosslinker activation agent with a target antigen at a
specific location on or in a sample of interest. As is well known
in the art, antibodies are glycoproteins belonging to the
immunoglobulin superfamily. Antibodies typically comprise two large
heavy chains and two small light chains, but various alternative or
modified antibody structures may be suitably employed in the
antibody reagents of the instant disclosure. For example, the
antibodies may be natural antibodies, artificial antibodies,
genetically engineered antibodies, monovalent antibodies,
polyvalent antibodies, monoclonal antibodies, polyclonal
antibodies, camelids, monobodies, single-chain variable fragments
(scFvs) and/or fragments or derivatives thereof, including Fab
fragments and F(ab')2 fragments. In certain applications, the
antibodies may be monospecific, polyspecific, humanized,
single-chain, chimeric, camelid single domain, shark single domain,
synthetic, recombinant, hybrid, mutated, CDR-grafted antibodies,
and/or fragments or derivatives thereof. In certain embodiments,
the antibodies may be derived from any suitable mammalian species.
For example, the antibodies may be derived from human, rat, mouse,
goat, guinea pig, donkey, rabbit, horse, llama, or camel. In other
embodiments, the antibodies may be derived from an avian species,
such as, for example, chicken or duck. The origin of the antibody
is defined by the genomic sequence, irrespective of the method of
production. The antibodies of the instant antibody reagents may be
of various isotypes, e.g., IgG, IgM, IgA, IgD, IgE or subclasses,
e.g., IgG1, IgG2, IgG3, IgG4. The antibodies may be produced
recombinantly, or by other means, which may include antibody
fragments that are still capable of binding an antigen, for
example, an Fab, an F(ab).sub.2, Fv, scFv, VhH, and/or V-NAR.
[0073] Suitable polyclonal antibodies for use in the instant
antibody reagents may be produced through a variety of methods. For
example, various animals may be immunized for this purpose by
injecting them with an antigen of interest, for example a target
biological molecule, or another molecule sharing an epitope of the
target biological molecule. Such antigen molecules may be of
natural origin or may be obtained by DNA recombination or synthetic
methods, or fragments thereof, and the desired polyclonal
antibodies may be obtained from the resulting sera and may be
purified. Alternatively, intact cells that array the target
biological molecule, or a suitable epitope of the target molecule,
may be used. Various adjuvants may also be used for increasing the
immune response to the administration of antigen, depending on the
animal selected for immunization. Examples of these adjuvants
include Freund's adjuvant, mineral gels such as aluminum hydroxide,
surfactant substances such as polyanions, peptides, oil emulsions,
haemocyanins, dinitrophenol, or lysolecithin.
[0074] Suitable monoclonal antibodies for use in the instant
antibody reagents may be obtained from hybridoma cells, which are
prepared by the fusion of spleen cells from an animal that has been
immunized with the desired antigen and myeloma cells. Cells
expressing the desired antibody are then identified by their
ability to bind the desired antigen. Stable hybridoma clones that
produce significant amounts of the desired antibody may then be
cultured to generate the antibody in useful amounts. These
techniques are well known in the art.
[0075] As noted above, in some embodiments, the instant antibody
reagents and detectable antibodies comprise an antibody specific
for a bridging antigen with high affinity. Suitable bridging
antigens for generating the high-affinity antibodies have been
described in detail above, including any molecule capable of
eliciting an immune response in a suitable animal or that can be
used to generate suitable antibodies by another means. These
include, for example, peptide antigens and non-peptidic,
small-molecule antigens, including biotin, digoxigenin,
dinitrophenyl, trinitrophenyl, and antigenic fluorophores. In order
to increase sensitivity and decrease background in amplified assays
using the instant antibody reagents, including detectable antibody
reagents, it is generally desirable to maximize the affinity and/or
specificity of each antibody reagent for its corresponding bridging
antigen. An antibody is therefore specific for a bridging antigen
with high affinity if it binds to the bridging antibody with high
affinity. As is understood by those of ordinary skill in the art,
affinities of antibodies for antigens are typically assessed using
an equilibrium parameter, the dissociation constant or "K.sub.D".
For a given concentration of antibody, the dissociation constant
roughly corresponds to the concentration of antigen at which half
the antibody is bound to an antigen and half the antibody is not
bound to an antigen. Accordingly, a lower dissociation constant
corresponds to a higher affinity of an antibody for the
antigen.
[0076] The dissociation constant is also related to the ratio of
the kinetic rate constants for dissociation and association of the
antibody and the antigen. Dissociation constants may therefore be
estimated either by equilibrium binding measurements or by kinetic
measurements. Such approaches are well known in the art. For
example, antibody-antigen binding parameters are routinely
determined from the kinetic analysis of sensorgrams obtained using
a Biacore surface plasmon resonance-based instrument (GE
Healthcare, Little Chalfont, Buckinghamshire, UK), an Octet
bio-layer interferometry system (Pall ForteBio Corp., Menlo Park,
Calif.), or the like. See, for example, U.S. Patent Application
Publication No. 2013/0331297 for a description of the determination
of dissociation constants for a series of antibody clones and their
corresponding peptide antigen binding partners.
[0077] Typical antibodies have equilibrium dissociation constants
in the range from micromolar to high nanomolar (i.e., 10.sup.-6 M
to 10.sup.-8 M). High affinity antibodies generally have
equilibrium dissociation constants in the lower nanomolar to high
picomolar range (i.e., 10.sup.-8 M to 10.sup.-10 M). Very high
affinity antibodies generally have equilibrium dissociation
constants in the picomolar range (i.e., 10.sup.-10 M to 10.sup.-12
M). Antibodies against peptides or other large molecules typically
have higher affinities (lower K.sub.Ds) for their antigens than
antibodies against small-molecule haptens, which may display
dissociation constants in the micromolar range or even higher.
[0078] The antibodies of the instant antibody reagents may be
optimized in order to increase their affinity for the bridging
antigen of the instant reagent compounds. For example, U.S. Patent
Application Publication No. 2013/0331297 discloses methods for
identifying antibody clones with high affinities that may be
suitably modified to generate the antibodies utilized in the
instant antibody reagents. In these methods, a short DNA fragment
encoding a synthetic peptide is fused to the heavy chains of the
gene pool encoding an antibody library of interest, and yeast cells
are transformed to generate a yeast display antibody library. The
yeast cells are screened with a high-speed fluorescence-activated
cell sorter (FACS) to isolate high-affinity antibody clones with
high specificity. Compared to other yeast display systems such as
Aga2, this system has an added advantage that the transformed yeast
cells secrete sufficient amounts of antibodies into the culture
medium to allow the culture media of the individual yeast clones to
be assayed directly to determine specificity and affinity of the
expressed antibodies, without requiring the additional steps of
cloning and antibody purification for identification of candidate
clones with the desired specificity and affinity.
[0079] The above-described yeast display library system makes use
of antibody libraries generated from immunized rabbits to produce
rabbit monoclonal antibodies with high specificity and affinity,
thus harnessing the superior ability of the rabbit immune system to
generate antibodies against small haptens or peptides with the
efficiency of yeast display to isolate antibody clones with
superior affinity and specificity. Using this approach, a panel of
rabbit monoclonal antibodies against small molecules, peptides, and
proteins was generated with antibody affinities in the range of
<0.01 to 0.8 nM. These affinities surpass the affinities of most
monoclonal antibodies from rodents generated using traditional
hybridoma technology. The approach also overcomes inherent issues
of low fusion efficiency and poor stability encountered with rabbit
hybridoma technology.
[0080] While the above-described yeast display library system is
one approach for optimizing binding affinities of the instant
antibody reagents, it should be understood that any suitable
approach may be used to optimize the affinities without limitation.
In some cases, suitable high-affinity antibodies may be available
without optimization.
[0081] Accordingly, in some embodiments, the antibody reagent or
detectable antibody is specific for the bridging antigen with a
dissociation constant of at most 100 nM, at most 30 nM, at most 10
nM, at most 3 nM, at most 1 nM, at most 0.3 nM, at most 0.1 nM, at
most 0.03 nM, at most 0.01 nM, at most 0.003 nM, or even lower. In
more specific embodiments, the antibody reagent or detectable
antibody is specific for the bridging antigen with a dissociation
constant of at most 1 nM, at most 0.3 nM, at most 0.1 nM, at most
0.03 nM, at most 0.01 nM, at most 0.003 nM, or even lower. In even
more specific embodiments, the antibody reagent or detectable
antibody is specific for the bridging antigen with a dissociation
constant of at most 100 pM, at most 30 pM, at most 10 pM, at most 3
pM, or even lower.
[0082] Examples of antibodies specific for a bridging antigen with
high affinity that are accordingly suitable for use in the instant
antibody reagents are described in U.S. patent application Ser. No.
15/017,626 and PCT International Application No. PCT/US2016/016913,
both of which were filed on Feb. 6, 2016, and both of which are
incorporated herein by reference in their entireties.
[0083] In another aspect, the instant disclosure further provides
antibody reagents capable of binding to target antigens other than
bridging antigens. In particular, these antibody reagents can
comprise an antibody specific for another target antigen, for
example a cellular marker or a cross-species antibody, and either a
crosslinker activation agent or a bridging antigen. In the case of
cellular markers, the target antigen may be a protein or other
antigenic molecule of interest either within a cell or on the
surface of a cell. The target antigen may in some cases be found
within a subcellular organelle, for example within the nucleus of a
cell or within the mitochondria. The target antigen may
alternatively be displayed on a surface of interest, such as, for
example, on an immunoblot or other type of two-dimensional medium.
The target antigen may in some cases be in impure form, in partly
purified form, or in purified form. In general, the target antigen
may be on or in any suitable surface, or may even be free in
solution, so long as it is available to interact specifically with
the antibody reagent. The antibody reagents ideally recognize the
target antigen with high specificity and selectivity and with low
background binding to non-target antigens.
[0084] Moreover, the target antigen of interest may be any protein
or other molecule of interest. In some embodiments, the target
antigen may be a cellular marker that provides information about
the disease state of a cell or tissue in an animal. For example,
the target antigen may be the estrogen receptor (ER), the HER2/neu
receptor (HER2), the progesterone receptor (PR), Ki-67, EGFR,
cytokeratin 1 (CK1), cytokeratin 5 (CK5), cytokeratin 6 (CK6),
cytokeratin 7 (CK7), cytokeratin 14 (CK14), cytokeratin 17 (CK17),
cytokeratin AE1/AE3, nestin, vimentin, ASMA, Ber-EP4, p16, p40,
p53, p63, c-kit, various CD markers, including those listed below,
or any other target antigen specifically recognizable by a primary
antibody. In some embodiments, multiple cellular markers may be
targeted. For example, in some embodiments, the target antigens may
be ER and PR. In other embodiments, the target antigens may be
HER2, ER, and PR or HER2, ER, and Ki-67. In still other
embodiments, the target antigens may be HER2, ER, PR, and Ki-67. In
yet still other embodiments, the target antigens may be Ki-67,
EGFR, and CK5. In even other embodiments, the target antigens may
be Ki-67, EGFR, CK5, and CK6.
[0085] In some embodiments, the target antigen may be CSF-1,
CSF-1R, CD163, VEGF, or a combination of these targets in a panel
(e.g., in an "M2" panel). In some embodiments, the target antigen
may be CD80, CD86, MHC Class II, or a combination of these targets
in a panel (e.g., in an "M1" panel). In specific embodiments, the
target antigen may be CD68, either alone or in combination with
another target antigen, for example in a panel, such as the above
panels. These target antigens are particularly useful in the
labeling and identification of macrophages in tissue samples.
[0086] Other specific target antigens include, without limitation,
4-1BB, AFP, ALK1, Amyloid A, Amyloid P, Androgen Receptor, Annexin
A1, ASMA, BCA225, BCL-1, BCL-2, BCL-6, BerEP4, Beta-Catenin,
Beta-HCG, BG-8, BOB-1, CA19-9, CA125, Calcitonin, Caldesmon,
Calponin-1, Calretinin, CAM 5.2, CD1a, CD2, CD3, CD4, CD5, CD7,
CD8, CD10, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD31,
CD33, CD34, CD38, CD42b, CD43, CD45 LCA, CD45RO, CD47, CD56, CD57,
CD61, CD68, CD79a, CD99, CD117, CD138, CD163, CDX2, CEA,
Chromogranin A, CMV, c-kit, c-MET, c-MYC, Collagen Type IV,
Complement 3c (C3c), COX-2, CXCR5, CK1, CK5, CK6, CK7, CK8, CK14,
CK18, CK17, CK19, CK20, CK903, CK AE1, CK AE1/AE3, D2-40, Desmin,
DOG-1, E-Cadherin, EGFR, EMA, ER, ERCC1, Factor VIII-RA, Factor
XIIIa, Fascin, FoxP1, FoxP3, Galectin-3, GATA-3, GATA-4, GCDFP-15,
GCET1, GFAP, GITR, Glycophorin A, Glypican 3, Granzyme B, HBME-1,
Helicobacter pylori, Hemoglobin A, Hep Par 1, HER-2, HHV-8, HMB-45,
HSV l/ll, ICOS, IFNgamma, IgA, IgD, IgG, IgM, IL17, IL4, Inhibin,
iNOS, Kappa Ig Light Chain, Ki-67, LAG-3, Lambda Ig Light Chain,
Lysozyme, Mammaglobin A, MART-1/Melan A, Mast Cell Tryptase, MLH1,
MOC-31, MPO, MSA, MSH2, MSH6, MUC1, MUC2, MUM1, MyoD1, Myogenin,
Myoglobin, Napsin A, Nestin, NSE, Oct-2, OX40, OX40L, p16, p21,
p27, p40, p53, p63, p504s, PAX-5, PAX-8, PD-1, PD-L1, Perforin,
PHH3, PIN-4, PLAP, PMS2, Pneumocystis jiroveci (carinii), PgR, PSA,
PSAP, RCC, S-100, SMA, SMM, Smoothelin, SOX10, SOX11, Surfactant
Apoprotein A, Synaptophysin, TAG 72, T-bet, TdT, Thrombomodulin,
Thyroglobulin, TIA-1, TIM3, TRAcP, TTF-1, Tyrosinase, Uroplakin,
VEGFR-2, Villin, Vimentin, WT-1, and the like.
[0087] In some embodiments, the instant antibody reagent may be a
cross-species reactive antibody reagent that is directed against
one or more sequences in an immunoglobulin molecule that do not
vary significantly between different immunoglobulins within the
same species. Such sequences are typically found within the
so-called "constant region" of the immunoglobulin sequence.
Recognition of these sequences is possible because the antibodies
used in these particular antibody reagents are generated by
immunization of a particular animal species, for example a goat,
with isolated immunoglobulins from a different animal species, for
example a mouse or a rabbit. An antibody generated in a goat
against a mouse immunoglobulin is thus referred to as a "goat
anti-mouse" antibody, and an antibody raised in a goat against a
rabbit immunoglobulin is thus referred to as a "goat anti-rabbit"
antibody. Polyclonal antibodies directed against a cross-species
immunoglobulin can be useful in signal amplification in an
immunologic assay due to their ability to recognize multiple
epitopes in the cross-species primary antibody.
[0088] It should be understood in the context of the instant
disclosure, that an antibody that binds to a cellular marker of
interest would typically be referred to as a "primary antibody" and
an antibody that binds to a cross-species immunoglobulin would
typically be referred to as a "secondary antibody". Such terms
should not be considered limiting in the context of the instant
disclosure, however, where multiple layers of antibody and antigen
may be employed in a single assay. The terms "primary antibody" and
"secondary antibody" in the instant disclosure should therefore be
considered limiting only as the terms are used in the claims to
distinguish one antibody from another.
[0089] In some antibody reagent embodiments, it may be advantageous
to attach additional phenol moieties, such as tyrosine moieties, or
other agents capable of reacting with the activated crosslinker
moiety of the instant reagent compounds, to the antibody reagents
of the instant disclosure, particularly to those comprising a
crosslinker activation agent. In this regard, it may be
particularly advantageous to conjugate polymers containing multiple
tyrosine moieties to an antibody-HRP conjugate. One skilled in the
art can immediately recognize that multiple tyrosines can be
incorporated into a variety of polymer constructs including but not
limited to peptide backbones, poly-tyrosine, and/or dendrimers.
[0090] As described above, the oxidation of tyrosine by HRP has
been exploited both to conjugate and to immobilize proteins. See
Minamihata et al. (2011) Bioconjugate Chem., 22, 2332 and Endrizzi
et al. (2006) Langmuir 22, 11305. Similar approaches find utility
in the instant amplified assay methods to increase the extent of
crosslinking of the activated crosslinker moieties of the instant
reagent compounds and thus to increase amplification of signal.
Moreover, the use of p-hydroxyphenylpropionic acid--casein or
p-hydroxyphenylpropionic acid--gelatin conjugates as blocking
agents has reportedly increased the sensitivity of labeling by TSA
in enzyme-linked immunosorbent assays (ELISAs), thus demonstrating
the utility of increasing levels of phenolic residues on labeling
in such methods. Bhattacharya et al. (1999) J. Immunol. Meth.
227:31; Bhattacharya et al. (1999) J. Immunol. Meth. 230:71.
[0091] In one embodiment, multiple tyrosines can be incorporated
into a linkable peptide and linked to an antibody-HRP conjugate. An
example of this approach is described below in the Examples
section. In other embodiments, linkable tyrosine polymers of a
variety of lengths can be prepared by initiating polymerization of
the N-carboxyanhydride of tyrosine using a bifunctional initiator
that includes an amine and a linkable moiety.
Oligonucleotide Reagents
[0092] As previously noted, the oligonucleotide reagents of the
instant disclosure generally comprise an oligonucleotide and a
crosslinker activation agent, where the oligonucleotide component
serves to associate the crosslinker activation agent with high
affinity to a complementary oligonucleotide. Crosslinker activation
agents suitably utilized in the instant oligonucleotide reagents,
as described above, include any agent capable of activating the
latent crosslinker moiety of a counterpart reagent compound in a
catalytic manner. Such agents include, for example, oxidase
enzymes, such as peroxidases, alkaline phosphatases, and glucose
oxidases. In preferred embodiments, the crosslinker activation
agent of the instant oligonucleotide reagents is a horseradish
peroxidase or a soybean peroxidase.
[0093] The oligonucleotide component of the oligonucleotide reagent
is chosen to be complementary to a target nucleic acid. In some
embodiments, the target nucleic acid is a bridging oligonucleotide,
for example, any of the bridging oligonucleotides in the
above-described reagent compounds. As will be described below, some
of the instant amplification methods involve multiple rounds of
amplification, and it may be desirable where an initial
amplification reaction makes use of a reagent compound containing a
bridging oligonucleotide for the immobilized bridging
oligonucleotides to be reacted with an oligonucleotide reagent that
targets that particular bridging oligonucleotide. In some
embodiments, the target nucleic acid is a genetic marker, for
example a genomic DNA sequence or an RNA sequence that has been
expressed in a cell. Hybridization of an oligonucleotide reagent
comprising a sequence complementary to the genetic marker thus
associates the crosslinker activation agent of the reagent to that
location.
Detectable Antibodies
[0094] In another aspect, the instant disclosure provides
detectable antibodies for use in the instant compositions, kits,
and methods. In particular, these antibodies comprise an antibody
specific for a bridging antigen with high affinity, for example as
in the above-described antibodies, and a detectable label. As would
be understood by those of ordinary skill in the art, the detectable
label of the detectable antibody should be capable of suitable
attachment to the antibody, and the attachment should be carried
out without significantly impairing the interaction of the antibody
with the bridging antigen.
[0095] In some embodiments, the detectable label may be directly
detectable, such that it may be detected without the need for any
additional components. For example, a directly detectable label may
be a fluorescent dye, a biofluorescent protein, such as, for
example, a phycoerythrin, an allophycocyanin, a peridinin
chlorophyll protein complex ("PerCP"), a green fluorescent protein
("GFP") or a derivative thereof (for example, a red fluorescent
protein, a cyan fluorescent protein, or a blue fluorescent
protein), luciferase (e.g., firefly luciferase, renilla luciferase,
genetically modified luciferase, or click beetle luciferase), or
coral-derived cyan and red fluorescent proteins (as well as
variants of the red fluorescent protein derived from coral, such as
the yellow, orange, and far-red variants), a luminescent species,
including a chemiluminescent species, an electrochemiluminescent
species, or a bioluminescent species, a phosphorescent species, a
radioactive substance, a nanoparticle, a SERS nanoparticle, a
quantum dot or other fluorescent crystalline nanoparticle, a
diffracting particle, a Raman particle, a metal particle, including
a chelated metal, a magnetic particle, a microsphere, an RFID tag,
a microbarcode particle, or a combination of these labels.
[0096] In other embodiments, the detectable label may be indirectly
detectable, such that it may require the employment of one or more
additional components for detection. For example, an indirectly
detectable label may be an enzyme that effects a color change in a
suitable substrate, as well as other molecules that may be
specifically recognized by another substance carrying a label or
that may react with a substance carrying a label. Non-limiting
examples of suitable indirectly detectable labels include enzymes
such as a peroxidase, an alkaline phosphatase, a glucose oxidase,
and the like. In specific embodiments, the peroxidase is a
horseradish peroxidase or a soybean peroxidase. Other examples of
indirectly detectable labels include haptens such as, for example,
a small molecule or a peptide. Non-limiting exemplary haptens
include nitrophenyl, dinitrophenyl, digoxygenin, biotin, a Myc tag,
a FLAG tag, an HA tag, an S tag, a Streptag, a His tag, a V5 tag, a
ReAsh tag, a FlAsh tag, a biotinylation tag, an Sfp tag, or another
chemical or peptide tag.
[0097] In specific embodiments, the detectable label is a
fluorescent dye. Non-limiting examples of suitable fluorescent dyes
may be found in the catalogues of Life Technologies/Molecular
Probes (Eugene, Oreg.) and Thermo Scientific Pierce Protein
Research Products (Rockford, Ill.), which are incorporated by
reference herein in their entireties. Exemplary dyes include
fluorescein, rhodamine, and other xanthene dye derivatives, cyanine
dyes and their derivatives, naphthalene dyes and their derivatives,
coumarin dyes and their derivatives, oxadiazole dyes and their
derivatives, anthracene dyes and their derivatives, pyrene dyes and
their derivatives, and BODIPY dyes and their derivatives. Preferred
fluorescent dyes include the DyLight fluorophore family, available
from Thermo Scientific Pierce Protein Research Products.
[0098] In some embodiments, the detectable label may not be
attached directly to the detectable antibody, but may be attached
to a polymer or other suitable carrier intermediate that allows
larger numbers of detectable labels to be attached to the antibody
than could normally be bound.
[0099] In specific embodiments, the detectable label is an
oligonucleotide barcode tag, for example the barcode tags disclosed
in PCT International Patent Publication No. WO2012/071428A2, the
disclosure of which is incorporated herein by reference in its
entirety. Such detectable labels are particularly advantageous in
immunoassays involving the isolation and/or sorting of targeted
samples, for example in flow cytometry-based multiplexed
immunodetection assays, and the like. These labels are also
advantageous in assays where the levels of target antigen in a
sample are low, and extreme sensitivity of detection is
required.
[0100] In some embodiments, the detectable antibodies of the
instant disclosure may comprise multiple detectable labels. In
these embodiments, the plurality of detectable labels associated
with a given detectable antibody may be multiple copies of the same
label or may be a combination of different labels that result in a
suitable detectable signal. In some embodiments, the detectable
antibodies are labeled with multiple probes having spectral
overlap. The use of such probes allows the different detectable
antibodies to be analyzed by spectral imaging techniques that, for
example, combine Fourier spectroscopy, charge-coupled device (CCD)
imaging, and optical microscopy to measure simultaneously in the
visible and near-infrared spectral range at all points in the
sample. Such techniques have been used, for example, in multicolor
spectral karyotyping (also known as "Sky imaging") of chromosomal
DNA in fluorescence in situ hybridization assays. Schrock et al.
(1996) Science 273:494. Similar approaches can be used with the
instant reagents and methods by suitable adaptation of the
detectable antibodies used in the instant methods.
Detectable Oligonucleotides
[0101] According to still another aspect, the instant disclosure
provides detectable oligonucleotides for use in the instant
compositions, kits, and methods. The detectable oligonucleotides
comprise an oligonucleotide and a detectable label. In specific
embodiments, the oligonucleotide is complementary to a bridging
oligonucleotide, including any of the bridging oligonucleotides
described above. As was the case with the detectable antibodies
described above, the detectable label of the detectable
oligonucleotides should be capable of suitable attachment to the
oligonucleotide, and the attachment should be carried out without
significantly impairing the interaction of the detectable
oligonucleotide with the complementary oligonucleotide. Any of the
labels described above for detectable antibodies may be suitably
adapted for use in the instant detectable oligonucleotides, as
would be understood by those of ordinary skill in the art.
Diagnostic Kits
[0102] In another aspect, the instant disclosure provides kits for
use in amplified assays for diagnostic or research purposes. The
diagnostic kits comprise one or more reagent compounds of the
instant disclosure, together with instructions for use in an assay.
In some embodiments, the kits further comprise an antibody reagent,
for example an antibody reagent that is specific for the bridging
antigen of a reagent compound at high affinity or an
oligonucleotide reagent, for example an oligonucleotide reagent
that is complementary to the bridging oligonucleotide of a reagent
compound. In some embodiments, the kits still further comprise a
detectable antibody specific for the bridging antigen of a reagent
compound at high affinity or a detectable oligonucleotide
complementary to the bridging oligonucleotide of a reagent
compound. Furthermore, it should be understood that the instant
kits may also comprise an antibody, or modified antibody, directed
at a cellular marker, so that the kit may be used in immunologic
assays for the detection of the cellular marker in a tissue sample,
in a suspension of cells, on another surface, or in another medium.
Likewise, the kits may comprise an oligonucleotide complementary to
a genetic marker. In some embodiments, the kits may comprise an
antibody, or modified antibody, directed at a cross-species
immunoglobulin, for example an anti-mouse antibody, an anti-rabbit
antibody, or the like. In these kits, the antibody reagent may be
used in immunologic assays for the detection of primary antibodies
of the target species. In some embodiments, the kits may comprise
one or more reagent compounds comprising a bridging oligonucleotide
and a latent crosslinker moiety. In some of these embodiments, the
kits further comprise a detectable oligonucleotide complementary to
the bridging oligonucleotide of the reagent compound.
[0103] In further embodiments, the kits may comprise further
components such as, for example, buffers of various compositions to
enable usage of the kit for staining cells or tissues, and cellular
counterstains to enable visualization of sample morphology. Kits
may be provided in various formats and include some or all of the
above listed components, or may include additional components not
listed here.
Methods for Signal Amplification
[0104] In another aspect, the instant disclosure provides methods
for signal amplification in biological assays that utilize the
reagent compounds and other associated reagents, including antibody
reagents and oligonucleotide reagents, disclosed herein. According
to some embodiments, these methods comprise the steps of: providing
a first sample that comprises a first target antigen, reacting the
first target antigen with a first antibody reagent specific for the
first target antigen, wherein the first antibody reagent comprises
a crosslinker activation agent, and reacting the first antibody
reagent with a first reagent compound, wherein the first reagent
compound is one of the reagent compounds described above, including
those comprising bridging antigens and those comprising bridging
oligonucleotides.
[0105] As shown in FIG. 1A, a basic immunologic assay, such as an
immunohistochemical assay, according to the instant methods can
include a traditional primary antibody reacting with a cellular
marker on a sample surface, as shown in step A. The bound primary
antibody, which represents the "target antigen" in this embodiment
of the method, is then reacted with an antibody reagent specific
for the cross-species primary antibody, as shown in step B. As
shown in this cartoon representation of the reaction, the antibody
reagent may comprise a cross-species antibody labeled with a
crosslinker activation agent, for example an HRP or other similar
enzyme (as represented in the drawing by a ribbon structure), as is
traditionally used in IHC staining. The bound antibody reagent can
then be treated with one of the instant reagent compounds, as shown
in step C, to amplify the signal output. The reagent compound in
this example comprises a bridging antigen (as represented in the
drawing by a triangle), and a latent crosslinker moiety, such as,
for example, tyramide. If an HRP or similar enzyme is used as the
crosslinker activation agent, this step would also include any
necessary coreactants, such as hydrogen peroxide or the like, as is
understood by those of ordinary skill in the art. The reaction
illustrated in step C of the drawing results in the labeling of
reactive groups in the vicinity of the cellular marker with
bridging antigens of the reagent compound. This step thus amplifies
the number of binding sites for subsequent reaction with a
detectable antibody, for example as shown in step D, where the
detectable antibody is specific for the bridging antigen of the
reagent compound. The detectable label associated with the
detectable antibody is indicated as a star in the drawing.
[0106] For example, a mouse primary antibody specific for a
cellular marker of interest may be used in the initial binding
step, as shown in step A of FIG. 1A, and a goat anti-mouse
secondary antibody, coupled to HRP or another crosslinker
activation agent, may be used in the second binding step, as shown
in step B of FIG. 1A, prior to treatment with a reagent compound,
as shown in step C of FIG. 1A. The same goat anti-mouse secondary
antibody reagent may be used with any mouse primary antibody, thus
minimizing the number of different antibody reagents and reagent
compounds required, as would be understood by those of ordinary
skill in the art.
[0107] As shown in the alternative method of FIG. 1B, the reagent
compound used in step C to react with the crosslinker activation
agent may alternatively comprise a bridging oligonucleotide (as
represented in the drawing by a wavy line), and a latent
crosslinker moiety, such as, for example, tyramide. As with the
method of FIG. 1A, this step results in the labeling of reactive
groups in the vicinity of the cellular marker, in this case with
bridging oligonucleotides of the reagent compound, and the
amplification of binding sites for subsequent reaction with a
detectable oligonucleotide, for example as shown in step D, where
the detectable oligonucleotide is complementary to the bridging
oligonucleotide of the reagent compound. Again, the detectable
label is indicated as a star in the drawing.
[0108] In further variations of the above methods, and as shown in
FIGS. 2A-2C, the target antigen may be a primary antibody labeled
with a bridging antigen (as illustrated by the two straight gray
bars in the drawings). The antibody reagent in these embodiments
thus comprises an antibody specific for the bridging antigen at
high affinity and a crosslinker activation agent. After binding to
a cellular marker on the sample, as shown in step A, the bridging
antigen-coupled primary antibody, as target antigen, is reacted as
shown in step B with an antibody reagent. Sensitivity is increased
in this approach due to the ability to attach multiple bridging
antigens to each primary antibody target. The subsequent steps are
typically the same as those shown in FIGS. 1A and 1B, with the
bound antibody reagent being reacted with a reagent compound, as
shown in step C, and with the bridging antigens (FIG. 2A) or
bridging oligonucleotides (FIG. 2B) that are attached in the
vicinity of the target antigen/cellular marker being labeled using
a detectable antibody or detectable oligonucleotide, as shown in
step D of these drawings.
[0109] FIG. 2C illustrates the multiplexing capability of the
instant methods, where a second primary antibody against a second
target antigen and labeled with a distinct bridging antigen (as
illustrated by the two wavy gray bars in the drawings) is reacted
with the sample in step D. The bound second primary antibody is
further reacted with a second antibody reagent that is specific for
the bridging antigen of the second primary antibody and that
comprises a crosslinker activation agent (step E). The bound second
antibody reagent is then reacted with a second reagent compound
comprising a bridging antigen and a latent crosslinker moiety (step
F). Not shown in this scheme is the subsequent labeling of the
amplified bridging antigens of the first and second reagent
compounds (triangles and circles) with detectable antibodies
specific for the different bridging antigens.
[0110] It will be understood by those of ordinary skill in the art
that it may be necessary in the multiplexed methods for the
crosslinker activation agent of the previous round of labeling to
be inactivated prior to addition of a subsequent round of reagent
compound. For example, in the method shown in FIG. 2C, the
crosslinker activation agent added at step B may need to be
inactivated prior to addition of the reagent compound in step F in
order to avoid background labeling by the second reagent compound
in the vicinity of the first target antigen/cellular marker.
Alternatively, if the crosslinker activation agent is attached to
its antibody through a cleavable linker, the crosslinker activation
agent may be releasable from the sample surface by a cleavage
reaction, as would be understood by those of ordinary skill in the
art. Inactivation or release of crosslinker activation agents after
each round of labeling in a multiplexed method greatly decreases
levels of background signal.
[0111] It should further be understood that signal amplification
methods involving a cross-species reactive antibody reagent (e.g.,
the methods shown in FIGS. 1A and 1B) can also be multiplexed by
suitable choice of primary antibody and cross-species reactive
secondary antibody pairs, but that the level of multiplexing
possible in such systems is significantly lower than the level
possible using primary antibodies labeled with bridging antigens as
shown in step A of FIGS. 2A-C.
[0112] The bridging antigens of the first and second reagent
compounds are illustrated in FIG. 2C as triangles and circles,
respectively. Although these bridging antigens should be
distinguishable from one other by their respective detectable
antibodies, it should also be understood that the bridging antigen
of the first reagent compound (triangle) can be the same as or
different from the bridging antigen used to label the first primary
antibody (straight gray bars). Likewise, the bridging antigen of
the second reagent compound (circle) can be the same as or
different from the bridging antigen used to label the second
primary antibody (wavy gray bars).
[0113] It should also be understood that corresponding multiplexed
methods are possible using reagent compounds comprising bridging
oligonucleotides and latent crosslinker moieties, for example by
substitution of the reagents used in steps C and F of FIG. 2C with
corresponding reagents comprising bridging oligonucleotides. It
should still further be understood that the number of different
target antigens and target nucleic acids detectable using
multiplexed methods with the instant compounds and reagents is
virtually unlimited due to the large range of structural
variability possible for the different bridging antigens and
bridging oligonucleotides.
[0114] The amplification possible using the instant methods is
readily apparent by reference to the graphic representations of
FIGS. 1A, 1B, 2A, and 2B, which illustrate the greatly increased
number of detectable antibodies and detectable oligonucleotides
that can be bound to a sample following amplification of the
bridging antigen or bridging oligonucleotide in step C of these
methods.
[0115] FIG. 3A shows yet another variant of the immunologic assay
methods that provides even higher levels of amplification,
specifically a two-round amplification method. The first three
steps in this approach are the same as were illustrated in FIG. 2A,
but instead of reacting the immobilized bridging antigens with a
detectable antibody, as shown in step D of FIG. 2A, they are
reacted with a second antibody reagent comprising an antibody
specific for the bridging antigen and a crosslinker activation
agent, preferably an HRP or similar enzyme. The second antibody
reagent can be the same as the first antibody reagent in this
method, if the bridging antigen used on the primary antibody of
step A was the same as used in the reagent compound of
amplification step C. In any case, the reagent compound used in
step E is preferably the same as the bridging antigen of the
reagent compound used in step C. This step immobilizes additional
bridging antigens in the vicinity of the target antigen. Subsequent
reaction of the immobilized bridging antigens with a detectable
antibody specific for the bridging antigen, as shown in step F of
FIG. 3A, results in a fully labeled sample. The large number of
bound labels (represented as stars in the drawing) graphically
illustrates the amplification potential of this method compared to
the one-round amplification approach outlined in FIG. 2A.
[0116] Shown in FIG. 3B is the counterpart method of FIG. 3A,
wherein the reagent compounds used in counterpart step C and/or
step E are compounds comprising a latent crosslinker moiety and a
bridging oligonucleotide. Where such reagent compounds are used in
one or more of the amplification steps, the subsequent steps make
use of a crosslinker activation agent that is coupled to an
oligonucleotide complementary to the amplified bridging
oligonucleotide (see step D). Not shown in FIG. 3B is the
subsequent reaction of the amplified bridging oligonucleotides with
complementary detectable oligonucleotides and the detection of the
amplified signal. This step would correspond to step D of FIGS. 1B
and 2B, as would be understood by those of ordinary skill in the
art.
[0117] A further variation in the second step of a two-step
amplification method is illustrated in FIG. 3C, where the amplified
bridging oligonucleotides associated with the sample surface in the
vicinity of a target antigen or target nucleic acid can serve as
primers for extension/replication reactions, thus resulting in
further amplification of detectable sequences. For example, as
shown in FIG. 3C, the bridging oligonucleotide may be complementary
to a sequence within a circular, single-stranded nucleic acid that
can serve as a template for a replicative reaction, such as rolling
circle replication (RCA). Extension of the bridging oligonucleotide
in the presence of a DNA polymerase and the necessary nucleotide
reagents generates a continuous linear replication of a complement
of the circular template nucleic acid. If the template is designed
so that the resulting linear replicated nucleic acid contains
appropriate complementary sequences, ideally repetitive sequences,
samples can be labeled with high sensitivity using detectable
oligonucleotides complementary to those sequences. Such approaches
have been used to visualize oligonucleotide probes in situ. See
Zhong et al. (2001) Proc. Nat'l Acad. Sci. U.S.A. 98:3940.
[0118] Other suitable replicative processes can be used to amplify
the sequence of a bound bridging oligonucleotide. Ideally such
replicative processes are isothermal amplification methods, so that
thermal cycling steps are not required in the amplification
process. See reviews by Kim et al. (2011) Bioanalysis 3:227 and
Zhao et al. (2015) Chem. Rev. 115:12491. Exemplary isothermal
replicative processes include without limitation loop-mediated
isothermal amplification (LAMP), strand displacement amplification
(SDA), helicase-dependent amplification (HDA), and nicking enzyme
amplification reaction (NEAR). Alternatively, non-enzymatic
methods, such as, for example, the hybridization chain reaction
(HCR), may be used to detect amplified bridging oligonucleotides,
where the bridging oligonucleotide serves as the "initiator" or
"trigger" sequence. See Ikbal et al. (2015) Trends Anal. Chem.
64:86 and Hauptmann et al. (2016) Methods 98:50. Such methods, and
others, can be readily adapted for the specific detection at high
sensitivity of bridging oligonucleotides specifically bound to a
sample surface in the vicinity of a target antigen or nucleic acid,
as would be understood by those of ordinary skill in the art.
Preferably, the amplification is a rolling circle amplification or
a hybridization chain reaction amplification.
[0119] In some embodiments according to this aspect of the
invention, the disclosure thus provides signal amplification
methods according to the following numbered paragraphs:
1. A method for signal amplification comprising: [0120] providing a
first sample comprising a first target antigen or a first target
nucleic acid; [0121] reacting the first target antigen with a first
antibody reagent or the first target nucleic acid with a first
oligonucleotide reagent, wherein the first antibody reagent
comprises an antibody specific for the first target antigen and a
crosslinker activation agent, and wherein the first oligonucleotide
reagent comprises an oligonucleotide complementary to the first
target nucleic acid and a crosslinker activation agent; [0122]
reacting the first antibody reagent or the first oligonucleotide
reagent with a first reagent compound, wherein the first reagent
compound comprises a bridging oligonucleotide and a latent
crosslinker moiety; and [0123] reacting the bridging
oligonucleotide with a first amplifiable oligonucleotide
complementary to the bridging oligonucleotide. 2. The method of
paragraph 1, wherein the first amplifiable oligonucleotide is
amplifiable by an isothermal amplification. 3. The method of
paragraph 2, wherein the isothermal amplification is a rolling
circle amplification. 4. The method of paragraph 2, wherein the
isothermal amplification is a hybridization chain reaction
amplification. 5. The method of any of paragraphs 1-4, further
comprising: [0124] amplifying the amplifiable oligonucleotide. 6.
The method of paragraph 5, further comprising: [0125] detecting the
amplified oligonucleotide.
[0126] In specific embodiments of the above methods, the
crosslinker activation agents of the instant antibody reagents and
oligonucleotide reagents comprise an enzyme. In more specific
embodiments, the enzyme is a peroxidase, an alkaline phosphatase,
or a glucose oxidase, and even more specifically is a peroxidase
such as HRP or soybean peroxidase.
[0127] As described above, the antibody reagents utilized in the
instant methods are preferably specific for bridging antigens with
a dissociation constant of at most 100 nM, at most 30 nM, at most
10 nM, at most 3 nM, at most 1 nM, at most 0.3 nM, at most 0.1 nM,
at most 0.03 nM, at most 0.01 nM, or at most 0.003 nM.
[0128] The detectable antibodies and detectable oligonucleotides of
the instant methods preferably comprise a detectable label. In
specific embodiments, the detectable label is a fluorophore, an
enzyme, an upconverting nanoparticle, a quantum dot, or a
detectable hapten. More specifically, the detectable label is a
fluorophore or is a peroxidase, an alkaline phosphatase, or a
glucose oxidase.
[0129] In specific embodiments, the instant methods further
comprise the step of detecting the detectable antibody or
detectable oligonucleotide.
[0130] In embodiments, the method of detection is an
immunohistochemical method. As described above, immunohistochemical
staining is widely used technique that is applied frequently to the
diagnosis of abnormal cells, such as tumor cells. Specific
molecular markers are characteristic of a particular tumor cell,
for example a breast cancer cell. IHC is also frequently used to
understand the distribution and localization of biomarkers and
differentially expressed proteins in different parts of a
biological tissue.
[0131] In specific embodiments, the target antigen is present
within a tissue section. Detection of antigens within tissue
sections is well understood by those of skill in the clinical
pathology arts. Exemplary methods of detecting antigens within a
tissue section are provided, for example, in Immunohistochemical
Staining Methods, 6.sup.th ed. (Dako/Agilent Technologies). It
should be understood that solid tissue samples, typically following
a fixation process, can be sectioned in order to expose one or more
target antigens of interest on the surface of the sample. The
analysis of consecutive tissue sections, i.e., sections that had
been adjacent, or nearly adjacent, to one another in the original
tissue sample, enables the recreation of a three-dimensional model
of the original tissue sample, or the increased capability for
multiplexing of target antigens, as will be described in more
detail below. In preferred embodiments, the first target antigen is
a cellular marker within a tissue section of a tumor sample.
[0132] In embodiments of the immunological assay methods where the
target antigen is present within a tissue section, the methods
preferably do not require the blocking of endogenous antigens
because the bridging antigen or bridging oligonucleotide is not
naturally present in the tissue. For comparison, with reagent
compounds comprising biotin or other antigens that occur naturally
in tissue samples, specific staining typically requires the
blocking of endogenous antigen by pretreatment of the tissue sample
with a blocking reagent in order to decrease background signals.
Such blocking steps are unnecessary where the bridging antigen or
bridging oligonucleotide of the reagent compound is not a molecule
that occurs naturally in a normal cell.
[0133] In other specific embodiments, the antigen detected by the
method is a cellular marker located in or on a cell. Such detection
is well understood, for example, by those of skill in the art of
cytometry. In some embodiments, the antigen may be on the surface
of a cell. In other embodiments, the antigen may be in the
cytoplasm of a cell. In still other embodiments, the antigen may be
in the nucleus of a cell. In some embodiments, the antigen may be
in more than one location in the cell.
[0134] The tissue analyzed according to the above methods may be
any suitable tissue sample. For example, in some embodiments, the
tissue may be connective tissue, muscle tissue, nervous tissue, or
epithelial tissue. Likewise, the tissue analyzed may be obtained
from any organ of interest. Non-limiting examples of suitable
tissues include breast, colon, ovary, skin, pancreas, prostate,
liver, kidney, heart, lymphatic system, stomach, brain, lung, and
blood.
[0135] In some embodiments, the detecting step is a fluorescence
detection step. Suitable fluorescence detection labels are
described in detail above.
[0136] In some embodiments, the method of detection further
comprises the step of sorting cells that have bound the detectable
antibody or detectable oligonucleotide. Cell sorting is a well
understood technique within the art of flow cytometry. Exemplary
flow cytometry methods of detection are provided, for example, in
Practical Flow Cytometry, 4.sup.th ed., Shapiro, Wiley-Liss, 2003;
Handbook of Flow Cytometry Methods, Robinson, ed., Wiley-Liss,
1993; and Flow Cytometry in Clinical Diagnosis, 4.sup.th ed., Carey
et al., eds, ASCP Press, 2007. The use of hydrazone-linked
antibody-oligonucleotide conjugates in quantitative multiplexed
immunologic assays, in particular, in quantitative flow cytometric
assays, is described in PCT International Publication No. WO
2013/188756 and in Flor et al. (2013) Chembiochem. 15:267-75.
[0137] It should be understood that the methods described herein
may be extended by repetition of the stain amplification steps
using primary antibodies having different specificities in order to
identify multiple cellular markers in or on a single sample. In
preferred embodiments, the different primary antibodies used in
subsequent steps are modified by bridging antigens or bridging
oligonucleotides, so that the primary antibodies can be recognized
by antibody reagents that are specific for the bridging antigens or
oligonucleotide reagents that are complementary to the bridging
oligonucleotides. The reagent compounds used in subsequent steps
are chosen to have latent crosslinker moieties appropriate for the
crosslinker activation agents used in the antibody reagents or
oligonucleotide reagents. Likewise, the bridging antigens and
bridging oligonucleotides of the subsequent reagent compounds are
chosen in view of the subsequent detection steps, for example
whether the bridging antigens or bridging oligonucleotides will be
detected by detectable antibodies or detectable oligonucleotides or
whether they will be recognized in further rounds of amplification
by other antibody reagents or oligonucleotide reagents.
[0138] In some embodiments, the multiplexing methods further
comprise reacting the bridging antigens or bridging
oligonucleotides with one or more of the detectable antibodies or
detectable oligonucleotides described above. In these embodiments,
the methods may further comprise detecting the detectable
antibodies or detectable oligonucleotides.
[0139] As described above, in some multiplexing method embodiments,
it may be beneficial to either remove or inactivate the crosslinker
activation agents of previous steps in order to avoid background
labeling in the subsequent steps. Methods to selectively strip
antibody reagents from their targets have been described in U.S.
patent application Ser. No. 15/017,626 and PCT International
Application No. PCT/US2016/016913. Methods to inactivate
crosslinker activation agents are known in the art. See Hauptmann
et al. (2016) Methods 98:50.
[0140] For example, in preferred embodiments, an antibody reagent
or oligonucleotide reagent, for example the antibody reagent shown
binding to bridging antigens in step B of FIGS. 2A and 2B, steps B
and E of FIG. 2C, and step D of FIG. 3A, or the oligonucleotide
reagent shown binding to bridging oligonucleotides in step D of
FIG. 3B, may be dissociated from the sample by a selective
treatment. Specifically, the selective treatment may dissociate the
antibody reagent or oligonucleotide reagent from the sample without
dissociating primary antibodies from the sample. More specifically,
the selective treatment may comprise treatment with a soluble
bridging antigen or a soluble bridging oligonucleotide. Such a
treatment may involve the use of relatively high concentrations of
the soluble bridging antigen or soluble bridging oligonucleotide,
for example at least 1 .mu.M, at least 10 .mu.M, at least 100
.mu.M, at least 1 mM, at least 10 mM, or even higher
concentrations, as would be understood by those of ordinary skill
in the art. In some embodiments the antibody reagent or
oligonucleotide reagent may be stripped by heating the sample
either alone or preferably with the above concentrations of soluble
bridging antigen or soluble bridging oligonucleotide.
[0141] It should also be understood that in the above methods, the
steps of dissociating the antibody reagent from the sample and
reacting the sample with an additional antibody reagent directed to
a primary antibody labeled with a different bridging antigen or
bridging oligonucleotide, amplification of the bound bridging
antigen or bound bridging oligonucleotide using a reagent compound
of the disclosure, and detection of the bridging antigen or
bridging oligonucleotide using a detectable antibody or detectable
oligonucleotide, may be repeated as many times as necessary in
order to detect the locations of as many target antigens on the
sample as desired. In some embodiments, the steps are repeated so
as to detect the location of at least three target antigens, at
least four target antigens, at least five target antigens, at least
ten target antigens, or even more target antigens on the sample. In
preferred embodiments, reaction of the amplified bridging antigens
or bridging oligonucleotides with detectable antibodies or
detectable oligonucleotides is not performed until all of the
different bridging antigens or bridging oligonucleotides have been
amplified, so that the different detectable antibodies or
detectable oligonucleotides can be added and detected in a single
step. In the multiplexed methods, the different detectable
antibodies or detectable oligonucleotides preferably comprise
different fluorophores, although the detectable antibodies and
detectable oligonucleotides may usefully comprise other detectable
labels that are suitably distinguishable from one another.
[0142] It should also be understood that the order of the steps
used in these assay methods may depend on the particular reaction
conditions used, and that additional reaction steps may also be
necessary to complete the assays in some cases. For example, if a
non-selective method is used to dissociate the antibody reagent
from the sample (e.g., heat, denaturation, etc.), it may be
necessary to include additional reaction steps in the assays.
Specifically, if the dissociation conditions also remove primary
antibodies from the sample, a further reaction with an additional
antibody specific for a cellular marker and labeled with a unique
bridging antigen or bridging oligonucleotide prior to reaction with
an additional antibody reagent specific for the binding antigen or
bridging oligonucleotide and an additional reagent compound may be
included in the process. In other words, the reaction of a new
primary antibody, secondary antibody reagent, and reagent compound
with a new target antigen will be included in the process for each
target antigen. In preferred embodiments, however, where the
antibody reagents are dissociated selectively, all of the desired
primary antibody reagents for reaction with all of the desired
target antigens may be added in an initial reaction step, and only
the antibody reagents specific for the different bridging antigens
or bridging oligonucleotides are added in subsequent cycles. Use of
selective treatments to dissociate antibody reagents from the
sample minimizes damage to the sample from harsh treatments and
therefore improves outcomes from the assays.
[0143] In the multiplexed assays, the methods may detect 2, 3, 5,
10, 20, 30, 50, 100, or even more different target antigens in a
single assay. Multiplexed immunohistochemical methods, including
their use in imaging and quantitation, have been reviewed recently.
See Stack et al. (2014) Methods 70:46.
[0144] In some embodiments, the instant methods of immunologic
assay comprise the analysis of adjacent or nearly adjacent sections
of a fixed tissue sample in order to increase the level of
multiplexing of target antigens possible for a given tissue sample
or to recreate a three-dimensional image of the sample. For
example, in some embodiments the methods may detect one or more
target antigens in serial sections of a tissue sample (i.e.,
sections that are adjacent, or nearly adjacent, to one another in
the sample). Such approaches are described in U.S. patent
application Ser. No. 15/017,626 and PCT International Application
No. PCT/US2016/016913.
[0145] It will be understood that the immunoassay of serial
sections of a given tissue sample provides for the greatly
increased multiplexing of antigen detection in view of current
hardware and software limitations. For example, although the
reagents and methods described herein in principle allow unlimited
multiplexing due to the unlimited variation in bridging antigens,
antibody reagents, and detectable antibodies, and bridging
oligonucleotides, oligonucleotide reagents, and detectable
oligonucleotides, such assays are nevertheless limited by the
number of fluorescent dyes that can currently be distinguished
simultaneously on a single tissue section with available detection
devices. Serial sections of the same tissue sample can, however, be
stained with different panels of primary antibodies to identify
different sets of target antigens by the reuse of the same panel of
detectable labels, for example fluorescent labels, on the different
sections.
[0146] It will also be understood that the immunoassay of serial
sections of a given tissue sample enables the analysis of target
tissue antigens in a third dimension, thus providing further
information regarding the overall structure of the sample tissue,
for example by tomographic techniques. In some embodiments, the
first sample and the second sample may not be serial sections of
the sample but may instead be separated in space within the
original tissue, thus providing still further information about the
relative spatial positioning of target antigens in the third
dimension. Those of ordinary skill in the art will understand the
utility of serial section images in the reconstruction of
three-dimensional tissue structures.
[0147] The reagent compounds and antibody reagents of the instant
disclosure may be usefully employed in a variety of immunochemical
methods of detection, including without limitation microscopic
imaging, pretargeting imaging, and other types of in vivo tumor and
tissue imaging, high content screening (HCS), immunocytochemistry
(ICC), immunomagnetic cellular depletion, immunomagnetic cell
capture, sandwich assays, general affinity assays, enzyme
immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot,
mass cytometry (CyTOF), arrays including microsphere arrays,
multiplexed microsphere array, microarray, antibody array, cellular
array, solution phase capture, lateral flow assays,
chemiluminescence detection, infrared detection, blotting methods,
including Western blots, Southwestern blot, dot blot, tissue blot,
and the like, or combinations thereof. Each of these assays may
benefit from the high level of amplification and multiplexing
achieved using the instant reagents.
[0148] The target antigens recognized using the instant methods may
be either polypeptide antigens, such as, for example, cellular
proteins of interest or other antibodies, or small-molecule
antigens, such as haptens. Other antigens may also be usefully
identified in the instant methods, as would be understood by those
of ordinary skill in the art. For example, targets identified using
the instant methods include proteins, microorganisms, viruses,
bacteria, drugs, hormones, toxins, biomolecules, lipids,
carbohydrates, nucleic acids, synthetic molecules, modified
proteins, and the like.
[0149] Although the methods described above are commonly applied to
immunologic assays for identifying target protein antigens, the
approaches and reagents of the instant disclosure can be readily
adapted for use in the assay and detection of nucleic acids. For
example, the reagent compounds described above can be used to
amplify signals in well-known assays for nucleic acids, for example
in in situ hybridization assays, such as fluorescence in situ
hybridization (FISH), and related techniques. Such techniques may
be used for the detection of any type of nucleic acid, including
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and any of
their natural or synthetic variants, without limitation. See, e.g.,
Volpi et al. (2008) BioTechniques 45:385 (doi 10.2144/000112811)
and Hauptmann et al. (2016) Methods 98:50.
[0150] For example, a two-pass tyramide signal amplification FISH
method has been used to detect single-copy genes in fixed bacterial
cells. See Kawakami et al. (2010) Microbes Environ. 25:15. In this
study, hapten-labeled oligonucleotide probes were hybridized with
the target nucleic acids under various conditions. The probes were
then detected using anti-hapten antibodies (e.g., anti-digoxigenin
antibodies) coupled to HRP in a first amplification step with
tyramide-DNP reagent compounds. Anti-DNP antibodies labeled with
HRP, together with tyramide-fluorophore reagent compounds, were
then used in a second amplification step to generate a fluorescent
signal for detection. The hybridization was optimized using probes
containing locked nucleic acids (LNAs). Other probes, such as
peptide nucleic acid (PNA) probes, were suggested as alternatives.
Stringency of hybridization was also optimized by modification of
binding conditions. The authors noted problematic non-specific
background signals, however, possibly due to the non-specific
binding of the HRP-labeled anti-digoxigenin antibodies to the
sample. Such non-specific binding can be readily overcome with the
instant reagents and methods by the choice of bridging antigen and
counterpart antibody or bridging oligonucleotide and counterpart
complementary oligonucleotide. Optimization of these reagent pairs
to increase their affinities for one another and to decrease their
non-specific binding can further reduce background signals.
[0151] Corresponding approaches using oligonucleotide probes
coupled to the above-described bridging antigens or bridging
oligonucleotides can be used to hybridize the bridging antigen or
bridging oligonucleotide to a target nucleic acid in a sample. As
with the just-described FISH technique, these samples can then be
treated with a reactive reagent that comprises a crosslinker
activation agent, for example HRP. Where the oligonucleotide probe
comprises a bridging antigen, the reactive reagent comprises an
antibody specific for the bridging antigen with high affinity, for
example, any of the antibody reagents described in detail above.
Where the oligonucleotide probe comprises a bridging
oligonucleotide, the reactive reagent comprises an oligonucleotide
complementary to the bridging oligonucleotide. In each case, the
reactive reagent is advantageously bound with higher affinity and
higher selectivity than was possible in the prior art, thus
improving sensitivity of the assays and decreasing background. The
samples are subsequently reacted, as already described, with
reagent compounds comprising a bridging antigen or a bridging
oligonucleotide and a latent crosslinker moiety, in order to react
with the bound crosslinker activation agent and thus crosslink
bridging antigens or bridging oligonucleotides in the vicinity of
the bound oligonucleotide probe at amplified levels. The
crosslinked bridging antigens or bridging oligonucleotides are then
either detected directly or subjected to a further amplification
step or steps, as previously described.
[0152] FIG. 4 illustrates an exemplary method, wherein different
oligonucleotide probes designed to target different sequences of a
target nucleic acid but labeled with the same bridging antigen
(designated by a triangle) are hybridized to the target nucleic
acid, as shown in step A. The target nucleic acid can be, for
example, an RNA molecule, as detected using single-molecule FISH
techniques. The hybridized probes are then reacted with an antibody
reagent specific for the bridging antigen with high affinity that
is labeled with a crosslinker activation agent (e.g., HRP), as
shown in step B. The bound antibody reagent is then reacted with a
reagent compound comprising a bridging antigen and a latent
crosslinker moiety (e.g., a tyramide-labeled bridging antigen), as
shown in step C. As mentioned above in the context of
immunohistochemical assays, the bridging antigen of the
oligonucleotide probes used in step A and the reagent compounds
used in step C can be the same or different, as desired. It should
also be understood that the bridging antigen can alternatively be
replaced with a bridging oligonucleotide, thus allowing reaction in
step B with a complementary oligonucleotide coupled to a
crosslinker activation agent. Likewise, the reagent compound used
in step C can alternatively comprise a bridging oligonucleotide
coupled to a latent crosslinker moiety and thus result in the
amplification of bridging oligonucleotides covalently associated
with the sample in the vicinity of the target nucleic acid. These
alternative method embodiments can be understood by analogy to the
immunohistochemical staining methods illustrated in step C of FIGS.
1B and 2B.
[0153] Also not shown in FIG. 4 is the reaction of the bridging
antigen with a detactable antibody, which can be understood by
analogy to step D of the methods illustrated in FIGS. 1A and 2A,
where the amplified bridging antigens resulting from step C are
labeled with a detectable antibody. Where the reactions generate
amplified bridging oligonucleotides, the labeling step can be
understood by analogy to step D of the methods illustrated in FIGS.
1B and 2B, where the amplified bridging oligonucleotides are
labeled with a detectable oligonucleotide.
[0154] FIG. 5 illustrates a variant of the method shown in FIG. 4,
where a target nucleic acid, for example in an in situ
hybridization assay, is reacted with multiple oligonucleotide
probes coupled to different bridging antigens or bridging
oligonucleotides. As shown in FIG. 5, the oligonucleotide probes
bound in step A are coupled to different bridging antigens
(designated by triangles and circles). The different bridging
antigens or oligonucleotides can accordingly be reacted with
different antibody or oligonucleotide reagents, for example as
shown in steps B and D, and different reagent compounds (containing
either bridging antigens or bridging oligonucleotides) can be
reacted sequentially by the bound crosslinker activation agents, as
shown in steps C and E. Not shown in this scheme is the labeling of
the amplified bridging antibodies with detectable antibodies. As
was the case in the method illustrated in FIG. 4, the target
nucleic acid in the method of FIG. 5 can be, for example, an RNA
molecule, as detected using single-molecule FISH techniques.
[0155] The method illustrated in FIG. 4 is particularly well suited
for single-molecule FISH and related technologies, where multiple,
relatively short, singly-labeled probes are hybridized with a given
target nucleic acid. See, for example, Raj et al. (2008) Nat.
Methods 5:877, which describes the simultaneous detection of
individual mRNA molecules within single cells stained with multiple
short oligonucleotide probes, each labeled with a single
fluorophore. The labeling of multiple probes with a limited number
of different labels, for example as illustrated in FIG. 5, may have
advantages where larger numbers of target nucleic acids need to be
detected simultaneously. See Levsky et al. (2002) Science 297:836,
for a method of expression profiling using "spectral barcodes". See
also Nederlof et al. (1990) Cytometry 11:126; Schrock et al. (1996)
Science 273:494; and Jakt et al. (2015) in In Situ Hybridization
methods, Neuromethods 99:509 (doi: 10.1007/978-1-4939-2303-8_27).
These and related techniques can be achieved using the compounds,
compositions, kits, and methods of the instant disclosure, in
particular according to the exemplary labeling methods illustrated
in FIGS. 4 and 5. Combination of spectral barcoding and
single-molecule FISH methodology can be particularly powerful. See
Kwon (2013) BMB Reports
(http://dx.doi.org/10.5483/bmbrep.2013.46.2.016). Commercial
resources are available for the design of suitable oligonucleotide
probes, for example in the design of Stellaris FISH probes. See,
for example, singlemoleculefish.com and www.biosearchtech.com. See
also Gaspar et al. (2015) WIREs Dev. Biol. 4:135 (doi:
10.1002/wdev.170) for recent review of quantitative single-molecule
RNA detection assays.
[0156] The instant antibody reagents, oligonucleotide reagents,
reagent compounds, and the just-described oligonucleotide probes
can accordingly be adapted for use in any nucleic acid detection
methods currently utilizing tyramide signal amplification
techniques, as would be understood by those of ordinary skill in
the art.
[0157] In some embodiments according to this aspect of the
invention, the disclosure thus provides signal amplification
methods according to the following numbered paragraphs:
1. A method for signal amplification comprising: [0158] providing a
first sample comprising a first target nucleic acid; [0159]
reacting the first target nucleic acid with a first oligonucleotide
probe, wherein the first oligonucleotide probe comprises an
oligonucleotide complementary to the first target nucleic acid and
a bridging antigen or a bridging oligonucleotide; and [0160]
reacting the first oligonucleotide probe with a first antibody
reagent or a first oligonucleotide reagent, wherein the first
antibody reagent comprises a crosslinker activation agent and an
antibody specific for the bridging antigen of the first
oligonucleotide probe with high affinity, and the first
oligonucleotide reagent comprises a crosslinker activation agent
and an oligonucleotide complementary to the bridging
oligonucleotide of the first oligonucleotide probe. 2. The method
of paragraph 1, wherein the crosslinker activation agent of the
first antibody reagent or the first oligonucleotide reagent
comprises an enzyme. 3. The method of paragraph 2, wherein the
enzyme is a peroxidase, an alkaline phosphatase, or a glucose
oxidase. 4. The method of paragraph 3, wherein the enzyme is a
peroxidase. 5. The method of paragraph 4, wherein the peroxidase is
a horseradish peroxidase or a soybean peroxidase. 6. The method of
paragraph 1, wherein the first target nucleic acid is a genetic
marker. 7. The method of paragraph 1, wherein the first target
nucleic acid is a bridging oligonucleotide. 8. The method of
paragraph 1, further comprising: [0161] reacting the first antibody
reagent or the first oligonucleotide reagent with a first reagent
compound, wherein the first reagent compound comprises a bridging
antigen or a bridging oligonucleotide and a latent crosslinker
moiety, including any of the reagent compounds described in detail
above. 9. The method of paragraph 8, further comprising: [0162]
reacting the bridging antigen of the first reagent compound with a
first detectable antibody specific for the bridging antigen or
reacting the bridging oligonucleotide of the first reagent compound
with a first detectable oligonucleotide complementary to the
bridging oligonucleotide. 10. The method of paragraph 9, wherein
the first detectable antibody or the first detectable
oligonucleotide comprises a detectable label. 11. The method of
paragraph 10, wherein the detectable label is a fluorophore, an
enzyme, an upconverting nanoparticle, a quantum dot, or a
detectable hapten. 12. The method of paragraph 11, wherein the
detectable label is a fluorophore. 13. The method of paragraph 11,
wherein the detectable label is a peroxidase, an alkaline
phosphatase, or a glucose oxidase. 14. The method of paragraph 9,
further comprising: [0163] detecting the first detectable antibody
or the first detectable oligonucleotide. 15. The method of
paragraph 8, further comprising: [0164] reacting the bridging
antigen or the bridging oligonucleotide of the first reagent
compound with a second antibody reagent or a second oligonucleotide
reagent, wherein the second antibody reagent comprises an antibody
specific for the bridging antigen of the first reagent compound and
a crosslinker activation agent, and the second oligonucleotide
reagent comprises an oligonucleotide complementary to the bridging
oligonucleotide of the first reagent compound and a crosslinker
activation agent. 16. The method of paragraph 15, wherein the
crosslinker activation agent of the second oligonucleotide reagent
comprises an enzyme. 17. The method of paragraph 16, wherein the
enzyme is a peroxidase, an alkaline phosphatase, or a glucose
oxidase. 18. The method of paragraph 17, wherein the enzyme is a
peroxidase. 19. The method of paragraph 18, wherein the peroxidase
is a horseradish peroxidase or a soybean peroxidase. 20. The method
of paragraph 15, further comprising: [0165] reacting the second
antibody reagent or the second oligonucleotide reagent with a
second reagent compound, wherein the second reagent compound
comprises a bridging oligonucleotide or a bridging oligonucleotide
and a latent crosslinker moiety, including any of the reagent
compounds described in detail above. 21. The method of paragraph
20, wherein the first reagent compound and the second reagent
compound comprise the same bridging antigen. 22. The method of
paragraph 20, wherein the first reagent compound and the second
reagent compound comprise the same bridging oligonucleotide. 23.
The method of paragraph 20, further comprising: [0166] reacting the
bridging antigen of the second reagent compound with a first
detectable antibody specific for the bridging antigen or reacting
the bridging oligonucleotide of the second reagent compound with a
first detectable oligonucleotide complementary to the bridging
oligonucleotide. 24. The method of paragraph 23, further
comprising: [0167] detecting the first detectable antigen or the
first detectable oligonucleotide.
[0168] In another variant of the above assays, rather than
attaching a bridging antigen or bridging oligonucleotide to the
oligonucleotide probe (or probes) used in the hybridization
reaction, the oligonucleotide probe is modified using a crosslinker
activation agent, for example a peroxidase enzyme. The reagent
therefore has a structure corresponding to the oligonucleotide
reagent shown in step D of FIG. 3B, except that the oligonucleotide
is designed to be complementary to a genetic marker target rather
than to a bridging oligonucleotide. The oligonucleotide will thus
target the crosslinker activation agent by hybridization to a
particular location in a sample, as would be understood in the art,
so that added reagent compounds, comprising a bridging antigen and
a latent crosslinker moiety or a bridging oligonucleotide and a
latent crosslinker moiety, upon activation by the crosslinker
activation agent, will be crosslinked to reactive groups in the
vicinity of the bound oligonucleotide probe.
[0169] The disclosure thus provides signal amplification methods
according to the following numbered paragraphs:
1. A method for signal amplification comprising: [0170] providing a
first sample comprising a first target nucleic acid; [0171]
reacting the first target nucleic acid with a first oligonucleotide
reagent, wherein the first oligonucleotide reagent comprises an
oligonucleotide complementary to the first target nucleic acid and
a crosslinker activation agent; and [0172] reacting the first
oligonucleotide reagent with a first reagent compound, wherein the
first reagent compound is any of the above-described reagent
compounds. 2. The method of paragraph 1, wherein the crosslinker
activation agent of the first oligonucleotide reagent comprises an
enzyme. 3. The method of paragraph 2, wherein the enzyme is a
peroxidase, an alkaline phosphatase, or a glucose oxidase. 4. The
method of paragraph 3, wherein the enzyme is a peroxidase. 5. The
method of paragraph 4, wherein the peroxidase is a horseradish
peroxidase or a soybean peroxidase. 6. The method of paragraph 1,
wherein the first target nucleic acid is a genetic marker. 7. The
method of paragraph 1, wherein the first target nucleic acid is a
bridging oligonucleotide. 8. The method of paragraph 1, further
comprising: [0173] reacting the bridging antigen of the first
reagent compound with a first detectable antibody specific for the
bridging antigen or reacting the bridging oligonucleotide of the
first reagent compound with a first detectable oligonucleotide
complementary to the bridging oligonucleotide. 9. The method of
paragraph 8, wherein the first detectable antibody or the first
detectable oligonucleotide comprises a detectable label. 10. The
method of paragraph 9, wherein the detectable label is a
fluorophore, an enzyme, an upconverting nanoparticle, a quantum
dot, or a detectable hapten. 11. The method of paragraph 10,
wherein the detectable label is a fluorophore. 12. The method of
paragraph 10, wherein the detectable label is a peroxidase, an
alkaline phosphatase, or a glucose oxidase. 13. The method of
paragraph 8, further comprising: [0174] detecting the first
detectable antibody or the first detectable oligonucleotide. 14.
The method of paragraph 1, further comprising: [0175] reacting the
bridging oligonucleotide of the first reagent compound with a
second oligonucleotide reagent, wherein the second oligonucleotide
reagent comprises an oligonucleotide complementary to the bridging
oligonucleotide of the first reagent compound and a crosslinker
activation agent; and [0176] reacting the second oligonucleotide
reagent with a second reagent compound comprising a bridging
oligonucleotide and a latent crosslinker moiety. 15. The method of
paragraph 14, wherein the crosslinker activation agent of the
second oligonucleotide reagent comprises an enzyme. 16. The method
of paragraph 15, wherein the enzyme is a peroxidase, an alkaline
phosphatase, or a glucose oxidase. 17. The method of paragraph 16,
wherein the enzyme is a peroxidase. 18. The method of paragraph 17,
wherein the peroxidase is a horseradish peroxidase or a soybean
peroxidase. 19. The method of paragraph 14, wherein the bridging
oligonucleotide of the first reagent compound and the bridging
oligonucleotide of the second reagent compound are the same. 20.
The method of paragraph 19, further comprising: [0177] reacting the
bridging oligonucleotide with a first detectable oligonucleotide
complementary to the bridging oligonucleotide. 21. The method of
paragraph 20, further comprising: [0178] detecting the first
detectable oligonucleotide.
[0179] In yet another variation of the above methods, amplification
of signal is improved by attaching additional groups to the
antibody reagent or oligonucleotide reagent that are reactive with
the activated form of the crosslinker moiety in order to increase
the number of crosslinks formed on the surface of the sample. For
example, FIG. 6 provides a schematic representation of an exemplary
two-step staining protocol for a cellular marker where the antibody
reagent contains added phenols for improved reaction with an
activated tyramide. In step A, the cellular marker is reacted with
an unlabeled primary antibody. In step B, the primary antibody is
reacted with a cross-species secondary antibody that has been
coupled to HRP (represented by ribbon diagrams) and a poly-tyrosine
peptide (represented by straight bars). In step C, the binding
signal is amplified by the addition of a reagent compound
comprising tyramide and a fluorophore (represented by starburst).
Specifically, catalytic activation of the reagent compound by HRP
results in the labeling of reactive groups in the vicinity of the
cellular marker, including the poly-tyrosine peptides. It should be
understood that the reagent compound used in step C of the method
could alternatively be a tyramide-modified bridging antigen or
bridging oligonucleotide, thus increasing the number of bridging
antigens or bridging oligonucleotides immobilized on the surface in
the vicinity of the antibody reagent by the reaction and thus the
ultimate signal. An example demonstrating the improved signal
obtained using this approach is provided below. See FIGS. 14A-14C.
PCT International Publication Number WO 2016/061460 describes the
use of oligo- or polymeric phenol-containing and/or phenylborate
containing substituents to enhance the amplification of signal in
TSA-based assays.
[0180] Any of the above methods find use in research and clinical
settings, without limitation. They may be used for diagnostic
purposes, including predictive screening and in other types of
prognostic assays, for example in a diagnostic laboratory setting
or for point of care testing. The methods may be used as companion
diagnostics during the course of a therapeutic treatment. The
methods are also well-suited for use in high-throughput
screens.
Methods of Preparation
[0181] In another aspect, the instant disclosure provides methods
of preparing the reagent compounds, antibody reagents, and
oligonucleotide reagents described above. In some embodiments, the
methods comprise the step of coupling an antibody or
oligonucleotide to a crosslinker activation agent using a chemical
coupling reaction. In specific embodiments, the antibody or
oligonucleotide and the crosslinker activation agent are coupled by
a high-efficiency conjugation moiety. In some embodiments the
methods comprise the steps of modifying an antibody or
oligonucleotide with a first conjugating reagent, modifying a
crosslinker activation agent with a second conjugating reagent, and
reacting the modified antibody or modified oligonucleotide with the
modified crosslinker activation agent to generate an antibody or
oligonucleotide reagent. In specific embodiments, the first
conjugating reagent and the second conjugating reagent associate
with one another at high efficiency.
[0182] In other embodiments, the methods comprise the step of
coupling a latent crosslinker moiety to a bridging antigen or
bridging oligonucleotide using a chemical coupling reaction. In
specific embodiments, the latent crosslinker moiety and the
bridging antigen or bridging oligonucleotide are coupled by a
high-efficiency conjugation moiety. In some embodiments the methods
comprise the steps of modifying a latent crosslinker moiety with a
first conjugating reagent, modifying a bridging antigen or bridging
oligonucleotide with a second conjugating reagent, and reacting the
modified latent crosslinker moiety with the modified bridging
antigen or bridging oligonucleotide to generate the reagent
compound. In specific embodiments, the first conjugating reagent
and the second conjugating reagent associate with one another at
high efficiency.
[0183] An example of this approach for preparing a reagent compound
is shown in FIG. 7, where tyramine (1) is reacted with an
acetal-protected succinimidyl 4-formyl benzoate (2) to produce a
tyramide 4-formyl benzoate compound (3). An N-terminal
aminooxy-modified bridging antigen or bridging oligonucleotide is
then reacted with compound 3 to form reagent compound 4, which
comprises a bridging antigen or bridging oligonucleotide coupled to
a tyramide residue through an oxime linkage.
[0184] FIG. 8 illustrates an alternative synthetic route for
preparing a reagent compound of the disclosure. Specifically,
tyramine (1) is reacted with succinic anhydride (5) to form acid
compound 6. This compound is coupled to an amino group of a
peptidic bridging antigen during the solid phase synthesis of the
bridging antigen using standard HBTU/EDC coupling conditions
followed by standard cleavage and deprotection steps to yield
reagent compound 7 with tyramine linked to the N-terminus of the
bridging antigen. The label can alternatively be attached to an
amine-functionalized oligonucleotide to yield reagent compound 7
with tyramine linked to the amine-functional group of the modified
oligonucleotide.
[0185] In other embodiments, the methods comprise the step of
coupling a crosslinker activation agent to an oligonucleotide probe
using a chemical coupling reaction. In specific embodiments, the
crosslinker activation agent and the oligonucleotide probe are
coupled by a high-efficiency conjugation moiety. In some
embodiments the methods comprise the steps of modifying a
crosslinker activation agent with a first conjugating reagent,
modifying an oligonucleotide probe with a second conjugating
reagent, and reacting the modified crosslinker activation agent
with the modified oligonucleotide probe to generate an
oligonucleotide probe reagent. In specific embodiments, the first
conjugating reagent and the second conjugating reagent associate
with one another at high efficiency.
[0186] Examples of related methods of preparation are provided in
U.S. patent application Ser. No. 15/017,626 and PCT International
Application No. PCT/US2016/016913
[0187] By high-efficiency, it is meant that the efficiency of
conversion of reactants to products is at least 50%, 70%, 90%, 95%,
or 99% complete under the conditions of the conjugation reaction.
In some embodiments, these efficiencies are achieved at no more
than 0.5 mg/mL, no more than 0.2 mg/mL, no more than 0.1 mg/mL, no
more than 0.05 mg/mL, no more than 0.02 mg/mL, no more than 0.01
mg/mL, or even lower concentrations of reactants.
[0188] The antibodies, oligonucleotide probes, bridging antigens,
latent crosslinker moieties, and crosslinker activation agents
usefully employed in the methods of preparation include any of the
examples described above. The first and second conjugating reagents
are chosen according to the desired outcomes. In particular,
high-efficiency conjugating reagents capable of specific and
selective reaction with amino or thiol groups are of particular
utility in the modification of peptides and proteins, such as
antibodies and peptidic bridging antigens. In addition, the first
and second conjugating reagents are chosen for their ability to
associate with one another at high efficiency, and thus to create
the high-efficiency conjugation moiety of some of the
above-described reagent compounds, antibody reagents, and
oligonucleotide probe reagents.
[0189] As described above, the resulting conjugation moiety may be
a covalent moiety or a non-covalent moiety, and the first and
second conjugating reagents used in the instant methods of
preparation are chosen accordingly. For example, in the case of a
non-covalent conjugation moiety, the first conjugating reagent
preferably comprises a selectively reactive group to attach the
reagent to particular reactive residues of a first molecule of
interest and a first component of the conjugation pair. Likewise,
the second conjugating reagent preferably comprises a selectively
reactive group to attach the reagent to particular reactive
residues of the second molecule of interest and a second component
of the conjugation pair. The first and second components of the
conjugation pairs are able to associate with one another
non-covalently at high efficiency and thus to generate the desired
product.
[0190] As previously described, examples of non-covalent
conjugation moieties include oligonucleotide hybridization pairs
and protein-ligand binding pairs. In the case of an oligonucleotide
hybridization pair, for example, the first molecule of interest
would be reacted with a first conjugating reagent that comprises
one member of the hybridization pair, and the second molecule of
interest would be reacted with a second conjugating reagent that
comprises the second member of the hybridization pair. The modified
molecules of interest can thus be mixed with one another, and the
association of the two members of the hybridization pair generates
the high-efficiency conjugation moiety.
[0191] Likewise, when a protein-ligand binding pair is used to
generate a non-covalent conjugation moiety, the first molecule of
interest is reacted with a first conjugating reagent that comprises
one or the other of the protein-ligand pair, and the second
molecule of interest is reacted with a second conjugating reagent
that comprises the complementary member of the protein-ligand pair.
The so-modified molecules of interest are then mixed with one
another to generate a high-efficiency conjugation moiety.
[0192] As was described in detail above, examples of
high-efficiency covalent conjugation moieties include hydrazones,
oximes, other Schiff bases, and the products of any of the various
click reactions. Exemplary hydrazino, oxyamino, and carbonyl
conjugating reagents for use in forming the high-efficiency
conjugation moieties are illustrated in U.S. Pat. No. 7,102,024 and
can be adapted for use in the instant reaction methods. As
described therein, the hydrazine moiety may be an aliphatic,
aromatic, or heteroaromatic hydrazine, semicarbazide, carbazide,
hydrazide, thiosemicarbazide, thiocarbazide, carbonic acid
dihydrazine, or hydrazine carboxylate. The carbonyl moiety may be
any carbonyl-containing group capable of forming a hydrazine or
oxime linkage with one or more of the above-described hydrazine or
oxyamino moieties. Preferred carbonyl moieties include aldehydes
and ketones. Activated versions of some of these reagents, for use
as conjugating reagents in the instant methods, are available
commercially, for example from Solulink, Inc. (San Diego, Calif.)
and Jena Bioscience GmbH (Jena, Germany). In some embodiments, the
reagents may be incorporated into a molecule of interest, for
example a bridging antigen, during its synthesis, for example
during the synthesis of a peptidic bridging antigen by solid phase
synthesis.
[0193] The incorporation of hydrazine, oxyamino, and carbonyl-based
monomers into oligonucleotides for use in immobilization and other
conjugation reactions is described in U.S. Pat. Nos. 6,686,461;
7,173,125; and 7,999,098. Hydrazine-based and carbonyl-based
bifunctional crosslinking reagents for use in the conjugation and
immobilization of biomolecules are described in U.S. Pat. No.
6,800,728. The use of high-efficiency bisaryl-hydrazone linkers to
form oligonucleotide conjugates in various detection assays and
other applications is described in PCT International Publication
No. WO 2012/071428. Each of the above references is hereby
incorporated by reference herein in its entirety.
[0194] Examples of novel conjugating reagents and conditions are
provided in U.S. patent application Ser. No. 15/017,626 and PCT
International Application No. PCT/US2016/016913. As described
therein, it should be understood that the relative orientation of
the different members of the conjugation moiety-forming groups on
the molecules of interest are generally not believed to be
important, so long as the groups are able to react with one another
to form the high-efficiency conjugation moiety.
[0195] The above-described conjugation methods provide several
advantages over traditional crosslinking methods, for example
methods using bifunctional crosslinking reagents. In particular,
the reactions are specific, efficient, and stable. The specificity
means that side reactions, such as homoconjugation reactions, do
not occur, or occur at extremely low levels. The efficiency means
that the reactions run to completion, or near completion, even at
low reactant concentrations, thus generating products in, or near,
stoichiometric amounts. The stability of the conjugation moieties
formed means that the resultant reagent compounds, antibody
reagents, and oligonucleotide probe reagents can be used for a wide
variety of purposes without concern that the conjugated products
will dissociate during use. In some cases, the above conjugation
methods allow the further advantage that the progress of the
conjugation reaction may be monitored spectroscopically, since in
some of the reactions a chromaphore is formed as the reaction
occurs.
[0196] The synthesis and stabilities of hydrazone-linked
adriamycin/monoclonal antibody conjugates are described in Kaneko
et al. (1991) Bioconj. Chem. 2:133-41. The synthesis and
protein-modifying properties of a series of aromatic hydrazides,
hydrazines, and thiosemicarbazides are described in U.S. Pat. Nos.
5,206,370; 5,420,285; and 5,753,520. The generation of
conjugationally-extended hydrazine compounds and fluorescent
hydrazine compounds is described in U.S. Pat. No. 8,541,555.
Alternative Binding Agents
[0197] In another aspect of the disclosure, the antibody or
oligonucleotide component of the instant antibody reagents may be
substituted with another agent capable of binding to target
antigens with high affinity. For example, aptamers are
single-stranded DNA or RNA oligomers that are capable of forming a
variety of tertiary structures and that are capable of binding to
targets such as metal ions, small molecules, proteins, viruses,
cells, and the like. See Ma et al. (2015) Chem. Soc. Rev. (DOI:
10.1039/C4CS00357H). Aptamers with high affinity and high
specificity for a given target molecule may be selected from a
random library using a procedure known as Sytematic Evolution of
Ligands by EXponential enrichment (SELEX), as is understood by
those of ordinary skill in the art. Once a suitable aptamer has
been identified and characterized, it may be further modified, for
example by the attachment of a label or other desired modification.
See, e.g., Wang et al. (2011) Curr. Med. Chem. 18:4175-4184 for a
review of aptamer-based fluorescent biosensors.
[0198] The reagents of the instant disclosure may therefore
advantageously employ aptamers, or other similar high-affinity and
high-selectivity binding agents, by coupling those agents with a
crosslinker activation agent, as described above for the antibody
reagents prepared from more traditional antibodies. For purposes of
this disclosure, it should therefore be understood that aptamers,
and other related high-affinity and high-selectivity binding
agents, should be considered to fall within the scope of the term
"antibody", as used and claimed herein, due to the ability of
aptamers to specifically recognize and bind specific target
molecules on a sample, as would be understood by those of ordinary
skill in the art. In some cases, an reagent comprising an aptamer
and a crosslinker activation agent will be referred to as an
"aptamer reagent".
Reagent Compositions
[0199] In another aspect, the disclosure provides reagent
compositions comprising an antibody reagent or oligonucleotide
reagent, as described in detail above, and a reagent compound, as
also described in detail above. The reagent compositions may be
provided in prepared forms, for example as a dried powder
containing both components of the composition. Most commonly,
however, the compositions are formed in solution, for example by
the addition of the reagent compound, either dry or in solution, to
a solution that already contains the antibody or oligonucleotide
reagent. The antibody reagent or oligonucleotide reagent is
preferably bound to a target antigen, bridging antigen, target
nucleic acid, or bridging oligonucleotide prior to the addition of
the reagent compound, so that activation of the latent crosslinker
moiety of the reagent compound by the crosslinker activation moiety
occurs in the vicinity of the target.
[0200] In some embodiments, the crosslinker activation agent of the
antibody reagent or oligonucleotide reagent comprises an enzyme,
for example, a peroxidase, an alkaline phosphatase, or a glucose
oxidase. In specific embodiments, the enzyme is a peroxidase, such
as a horseradish peroxidase or a soybean peroxidase.
[0201] In some embodiments, the antibody of the antibody reagent is
specific for a bridging antigen with high affinity. The antibody
reagent may be specific for any of the above-described antigens,
including biotin and small-molecule haptens. More specifically, the
antibody reagent may be specific for a bridging antigen comprising
a peptide. In some embodiments, the antibody is specific for a
bridging antigen comprising a plurality of antigenic determinants,
for example, a bridging antigen wherein each antigenic determinant
in the plurality of antigenic determinants is the same or wherein
the plurality of antigenic determinants comprises a linear
repeating structure. More specifically, the linear repeating
structure may comprise a linear repeating peptide structure. In
some embodiments, the plurality of antigenic determinants may
comprise at least three antigenic determinants. In some
embodiments, the bridging antigen may comprise a branched
structure. In some embodiments, the first antibody reagent may be
specific for a bridging antigen comprising a peptide comprising a
non-natural residue, such as a non-natural stereoisomer or a
.beta.-amino acid.
[0202] In some embodiments, the antibody of the antibody reagent is
specific for a bridging antigen with a dissociation constant of at
most 100 nM, at most 30 nM, at most 10 nM, at most 3 nM, at most 1
nM, at most 0.3 nM, at most 0.1 nM, at most 0.03 nM, at most 0.01
nM, or at most 0.003 nM.
[0203] In some embodiments, the antibody of the antibody reagent is
specific for a cellular marker such as, for example, a cellular
marker is selected from the group consisting of: 4-1BB, AFP, ALK1,
Amyloid A, Amyloid P, Androgen Receptor, Annexin A1, ASMA, BCA225,
BCL-1, BCL-2, BCL-6, BerEP4, Beta-Catenin, Beta-HCG, BG-8, BOB-1,
CA19-9, CA125, Calcitonin, Caldesmon, Calponin-1, Calretinin, CAM
5.2, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20,
CD21, CD22, CD23, CD25, CD30, CD31, CD33, CD34, CD38, CD42b, CD43,
CD45 LCA, CD45RO, CD47, CD56, CD57, CD61, CD68, CD79a, CD80, CD86,
CD99, CD117, CD138, CD163, CDX2, CEA, Chromogranin A, CMV, c-kit,
c-MET, c-MYC, Collagen Type IV, Complement 3c (C3c), COX-2, CXCR5,
CK1, CK5, CK6, CK7, CK8, CK14, CK18, CK17, CK19, CK20, CK903, CK
AE1, CK AE1/AE3, CSF-1, CSF-1R, D2-40, Desmin, DOG-1, E-Cadherin,
EGFR, EMA, ER, ERCC1, Factor VIII-RA, Factor XIIIa, Fascin, FoxP1,
FoxP3, Galectin-3, GATA-3, GATA-4, GCDFP-15, GCET1, GFAP, GITR,
Glycophorin A, Glypican 3, Granzyme B, HBME-1, Helicobacter pylori,
Hemoglobin A, Hep Par 1, HER2, HHV-8, HMB-45, HSV l/ll, ICOS,
IFNgamma, IgA, IgD, IgG, IgM, IL17, IL4, Inhibin, iNOS, Kappa Ig
Light Chain, Ki-67, LAG-3, Lambda Ig Light Chain, Lysozyme,
Mammaglobin A, MART-1/Melan A, Mast Cell Tryptase, MHC Class II,
MLH1, MOC-31, MPO, MSA, MSH2, MSH6, MUC1, MUC2, MUM1, MyoD1,
Myogenin, Myoglobin, Napsin A, Nestin, NSE, Oct-2, OX40, OX40L,
p16, p21, p27, p40, p53, p63, p504s, PAX-5, PAX-8, PD-1, PD-L1,
Perforin, PHH3, PIN-4, PLAP, PMS2, Pneumocystis jiroveci (carinii),
PR, PSA, PSAP, RCC, S-100, SMA, SMM, Smoothelin, SOX10, SOX11,
Surfactant Apoprotein A, Synaptophysin, TAG 72, T-bet, TdT,
Thrombomodulin, Thyroglobulin, TIA-1, TIM3, TRAcP, TTF-1,
Tyrosinase, Uroplakin, VEGF, VEGFR-2, Villin, Vimentin, and
WT-1.
[0204] In some embodiments, the antibody of the antibody reagent is
a cross-species antibody.
[0205] In some embodiments, the oligonucleotide of the
oligonucleotide reagent is complementary to a bridging
oligonucleotide, more specifically the bridging oligonucleotide of
the reagent compound. In other embodiments, the oligonucleotide of
the oligonucleotide reagent is complementary to a genetic
marker.
[0206] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein may be made without
departing from the scope of the invention or any embodiment
thereof. Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
Examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention.
Examples
Synthesis of Tyramide-Modified Compounds
Experimental
[0207] Synthesis of reagent compounds comprising peptidic bridging
antigens with tyramide at their amino termini was performed using
the following methods. See also FIGS. 7 and 8 for reaction
schemes.
[0208] 4-Dimethoxymethylbenzoic acid: 4-Formylbenzoic acid (20 g;
0.133 mmol; SigmaAldrich, St. Louis, Mo.) was dissolved in MeOH
(125 mL) followed by the addition of trimethylorthoformate (16.1
mL; 0.147 mmol) and 4 N HCl/dioxane (2.0 mL; SigmaAldrich). The
reaction was stirred at room temperature for 5 h. The solvent was
removed on the rotavap and the product was used without further
purification.
[0209] Succinimidyl 4-Dimethoxymethylbenzoate (1.57 g; 8.02 mmol)
was dissolved in DCM (75 mL), added N-hydroxysuccinimide (0.92 mg;
8.02 mmol, SigmaAldrich, St. Louis, Mo.) and EDC (2.31 g; 12.0
mmol; Oakwood Chemical). The heterogeneous reaction mixture was
stirred at room temperature for 3 h. TLC (100% EtOAc) indicated
that the reaction was >90% complete. To the reaction mixture was
added a solution of tyramine (1.10 g; 8.02 mmol) in DMF (30 mL).
The reaction mixture was stirred overnight at room temperature. The
reaction mixture was concentrated to dryness on the rotavap and the
oily residue was dissolved in DCM (100 mL) washed with aqueous
saturated bicarbonate (2.times.100 mL) and brine (100 mL). The
organic phase was dried over magnesium sulfate, filtered and
concentrated to give a pale brown oil with some solids. The oil was
dissolved in ethyl acetate (.about.10 mL) and placed in the freezer
overnight. On incubation at 4.degree. C. more solids precipitated.
The solids were isolated by filtration to give 675 mg of a white
solid. The purity and structure of the product was confirmed by
NMR.
[0210] To a solution of tyramine (58 mg; 0.42 mmol) in DMF (4.91
mL) was added a solution of succinic anhydride (42.3 mg; 0.42
mmol), TEA (90 .mu.L; 0.63 mmol) and DMAP (few crystals). The
reaction was stirred at room temperature for two hours. TLC
(DCM/MeOH (9/1; visualization UV and ninhydrin) indicated complete
conversion to product. The solution was used directly in solid
phase peptide synthesis.
[0211] Table 2 lists the tyramide-bridging antigen reagent
compounds prepared by solid-phase peptide synthesis using the
tyramide acid shown as compound 6 in FIG. 8 (synthesized at
Innopep, Inc, San Diego, Calif. (www.innopep.com)). All peptides
were >95% pure and had the expected molecular weights by mass
spectrometry. The rabbit monoclonal antibodies and their
corresponding bridging antigens have been shown to display
dissociation constants in the range from about 10 pM to about 200
pM. See also U.S. patent application Ser. No. 15/017,626 and PCT
International Application No. PCT/US2016/016913.
TABLE-US-00002 TABLE 2 Tyramide-labeled peptidic bridging antigen
sequences Peptide Sequence PEP6 Tyr-ETSGLQEQRNHLQGK-CONH2 (SEQ ID
NO: 1) PEP7 Tyr-GAPGKKRDMSSDLERD-NH2 (SEQ ID NO: 2) PEP1
Tyr-LALQAQPVPDELVTK-COOH (SEQ ID NO: 3) PEP5
Tyr-RPHFPQF-pY-SASGTA-NH2 (SEQ ID NO: 4)
Preparation of Tyramide-Labeled 3.lamda.-Tandem Repeat Bridging
Antigen Peptides
[0212] A 1 mg/mL solution of tyramide-4FBAA (compound (3) in FIG.
7) in MeOH was prepared. The AOA-peptide (100 .mu.g) was dissolved
in 100 mM MES, pH 5.0 (50 .mu.L). To this solution was added MeOH
(50 .mu.L--volume of AOA-peptide/MeOH solution), as shown in the
second step of FIG. 7. Added tyramide-4FBAA/MeOH solution
containing 1.1 equivalents tyramide 4FBAA. The reaction mixture was
incubated overnight at room temperature. The reaction mixture was
used without further purification.
[0213] Table 3 shows the sequence of a 3.lamda.-tandem repeat
peptide used to form tyramide-labeled reagent compounds.
TABLE-US-00003 TABLE 3 3X-Tandem repeat peptide sequence Peptide
Sequence PEP5-3X AOA-Peg2-RPHFPQF-pY-SASGTARPHFPQF-pY-
SASGTARPHFPQF-pY-SASGTA-OH (SEQ ID NO: 5)
Fluorescence Staining and Imaging
[0214] The following tyramide-based protocols were employed in the
below-described immunofluorescence staining experiments. The slides
were imaged on a Vala Sciences IC200Hist Imager (Vala Sciences, San
Diego, Calif.). The images were processed using open source ImageJ
software.
[0215] Unless otherwise indicated all breast cancer tissue was
purchased from ILSBio (www.ilsbio.com).
[0216] Following are the protocols employed for the various
tyramide-based staining experiments.
Manual Staining Protocol:
Antigen Retrieval
[0217] 1. Slides were dewaxed as follows:
TABLE-US-00004 [0217] Xylene 5 min Xylene 5 min 100% Ethanol 2 min
100% Ethanol 2 min 95% Ethanol 2 min
[0218] 2. Wash 2.times. with tap water 2 min each. [0219] 3. Wash
1.times. with distilled water 2 min. [0220] 4. Antigen retrieval
was accomplished by steaming in 10 mM citric acid pH 6.0 for 15
min. [0221] 5. Slides were cooled in pressure cooker for 10 min
before releasing pressure. [0222] 6. Pressure was released and
slides wer moved to hot distilled water for 2 min. [0223] 7. Slides
were washed under running tap water for 5 min. [0224] 8. Slides
were rinsed in wash buffer for 5 mins. [0225] 9. Circles were drawn
around the tissue using a hydrophobic pen. [0226] 10. Slides were
blocked with normal serum (3% goat or rabbit serum, sometimes other
serum depending on stain) for 20 min.
[0227] Protocol A--tyramide-fluorophore staining: Following antigen
retrieval the following steps were employed to stain with
tyramide-fluorophore reagents: [0228] 1. After removal of previous
solution, 150 .mu.L to 200 .mu.L of a 0.5 ng/.mu.L Ki-67 (BD
Biosciences, San Diego, Calif.) primary antibody were added
directly on slide, which can be diluted using antibody diluent, and
incubated for 1 hr at room temperature. [0229] 2. Slides were
washed 3.times. with wash buffer for 5 min each. [0230] 3. To the
slide was added the anti-mouse HRP (Cell IDx, San Diego, Calif.) at
10 ng/.mu.L for 30 minutes. [0231] 4. Slides were washed 3.times.
in wash buffer for 5 min each. [0232] 5. To the slide was added the
tyramide-Dy650 (Dyomics GmbH, Jena, Germany) at 50 .mu.g/mL for 10
minutes. [0233] 6. Slides were washed 3.times. in wash buffer for 5
min each. [0234] 7. Slides were rinsed with distilled water,
removing excess water with paper towel. [0235] 8. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0236] Protocol B-- tyramide-hapten staining: Following antigen
retrieval the following steps were employed to stain with
tyramide-hapten, e.g., digoxigenin, reagents: [0237] 1. Anti-Ki-67
at 0.5 ng/mL, 150 uL, was added directly on the slide and incubated
for 1 h. [0238] 2. The slide was washed 3.times. in wash buffer for
5 min each. [0239] 3. To the slide was added the
tyramide-digoxigenin at 50 .mu.g/mL for 10 minutes.
[0239] ##STR00001## [0240] 4. Slides were washed 3.times. in wash
buffer for 5 min each. [0241] 5. To the slide was added the
anti-digoxigenin-Dy650 (Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L
for 10 minutes [0242] 6. Slides were washed 3.times. in wash buffer
for 5 min each. [0243] 7. Slides were rinsed with distilled water,
removing excess water with paper towel. [0244] 8. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0245] Protocol C-- one-round tyramide-peptide bridging antigen
staining: Following antigen retrieval the following steps were
employed to stain with secondary antibody-HRP
polymer/tyramide-peptide bridging antigen/fluorophore-labeled
anti-peptide antibody three step protocol: [0246] 1. Steps 1-4 of
Protocol A were followed using Ki-67 as primary antibody. [0247] 2.
To the slide was added the tyramide-PEP6 at 50 .mu.g/mL for 10
minutes. [0248] 3. Slides were washed 3.times. in wash buffer for 5
min each. [0249] 4. To the slide was added the anti-PEP6-Dy650
(Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L for 10 minutes. [0250]
5. Slides were washed 3.times. in wash buffer for 5 min each.
[0251] 6. Slides were rinsed with distilled water, removing excess
water with paper towel. [0252] 7. 1-3 drops of Fluoroshield with
DAPI (Immunobiosciences, Inc, cat# AR-6501-01) was added to each
slide and after 3-5 min in the dark at room temperature the
coverslip was applied.
[0253] Protocol D--one-round tyramide-biotin staining: Following
antigen retrieval the following steps were employed to stain with
secondary antibody-HRP polymer/tyramide-biotin/fluorophore-labeled
streptavidin three step protocol: [0254] 1. After removal of
previous blocking solution block the slides with a 0.05% solution
of Streptavidin in PBS. [0255] 2. Slides were washed 2.times. in
wash buffer for 5 min each. [0256] 3. Block the slide with a 0.005%
solution of Biotin in PBS. [0257] 4. Slides were washed 2.times. in
wash buffer for 5 min each. [0258] 5. After removal of previous
solution, 150 .mu.L to 200 .mu.L of a 100 pg/.mu.L Ki-67 (BD
Biosciences, San Diego, Calif.) primary antibody was added directly
on slide, which can be diluted using antibody diluent, and
incubated for 1 hr at room temperature. [0259] 6. Slides were
washed 3.times. with wash buffer for 5 min each. [0260] 7. To the
slide was added the anti-mouse HRP (Cell IDx, San Diego, Calif.) at
10 ng/.mu.L for 30 minutes. [0261] 8. Slides were washed 3.times.
in wash buffer for 5 min each. [0262] 9. To the slide was added the
tyramide-biotin (Cell IDx, San Diego, Calif.) at 50 .mu.g/mL for 10
minutes. [0263] 10. Slides were washed 3.times. in wash buffer for
5 min each. [0264] 11. To the slide was added the
streptavidin-Dy650 (Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L for
10 minutes. [0265] 12. Slides were washed 3.times. in wash buffer
for 5 min each. [0266] 13. Slides were rinsed with distilled water,
removing excess water with paper towel. [0267] 14. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0268] Protocol E--two-round tyramide-biotin staining: The
following protocol was used for two-round staining with secondary
antibody-HRP polymer/tyramide biotin. [0269] 1. Steps 1-10 in
Protocol D were followed using Ki-67 as primary antibody. [0270] 2.
To the slide was added the streptavidin-HRP (Cell IDx, San Diego,
Calif.) at 10 ng/.mu.L for 10 minutes. [0271] 3. Repeat steps 8-10
in Protocol D. [0272] 4. To the slide was added the
streptavidin-Dy650 (Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L for
10 minutes. [0273] 5. Slides were washed 3.times. in wash buffer
for 5 min each. [0274] 6. Slides were rinsed with distilled water,
removing excess water with paper towel. [0275] 7. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0276] Protocol F-- two-round tyramide-peptide bridging antibody
staining: The following protocol was used for two-round staining
with secondary antibody-HRP polymer/tyramide-peptide. [0277] 1.
After antigen retrieval, Protocol A steps 1-4 were performed.
[0278] 2. To the slide was added tyramide-PEP6 and incubated at 50
ng/mL for 10 minutes. [0279] 3. The slide was washed 3.times. with
wash buffer for 5 min each. [0280] 4. To the slide was added
150-200 uL of anti-PEP6-HRP and incubated for 10 minutes. [0281] 5.
The slide was washed 3.times. with wash buffer for 5 min each.
[0282] 6. To the slide was added tyramide-PEP6 and incubated at 50
ng/mL for 10 minutes. [0283] 7. The slide was washed 3.times. with
wash buffer for 5 min each. [0284] 8. To the slide was added the
anti-PEP6-Dy650 (Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L for 10
minutes [0285] 9. Slides were washed 3.times. in wash buffer for 5
min each. [0286] 10. Slides were rinsed with distilled water,
removing excess water with paper towel. [0287] 11. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0288] Protocol G--one-round primary antibody-peptide bridging
antigen/tyramide-peptide bridging antigen staining: The following
protocol was used for one-round staining with primary
antibody-peptide bridging antigen/secondary antibody-HRP
polymer/tyramide-peptide bridging antigen. [0289] 1. Following
antigen retrieval, 150 .mu.L to 200 .mu.L of a 10 pg/.mu.L
anti-HER2-PEP5 (Cell IDx, San Diego, Calif.) primary antibody was
added directly on slide, which was diluted using antibody diluent,
and incubated for 1 hr at room temperature. [0290] 2. Slides were
washed 3.times. with wash buffer for 5 min each. [0291] 3. To the
slide was added the anti-PEP5-HRP at 10 ng/.mu.L for 30 minutes.
[0292] 4. Slides were washed 3.times. in wash buffer for 5 min
each. [0293] 5. To the slide was added the tyramide-PEP6 at 50
.mu.g/mL for 10 minutes. [0294] 6. Slides were washed 3.times. in
wash buffer for 5 min each. [0295] 7. To the slide was added the
anti-PEP6-Dy650 (Dyomics GmbH, Jena, Germany) at 5 .mu.g/mL for 10
minutes. [0296] 8. Slides were washed 3.times. in wash buffer for 5
min each. [0297] 9. Slides were rinsed with distilled water,
removing excess water with paper towel. [0298] 10. 1-3 drops of
Fluoroshield with DAPI (Immunobiosciences, Inc, cat# AR-6501-01)
was added to each slide and after 3-5 min in the dark at room
temperature the coverslip was applied.
[0299] Protocol H-- two-round primary antibody-peptide bridging
antigen/tyramide-peptide bridging antigen staining: The following
protocol was used for two-round staining with primary
antibody-peptide bridging antigen/secondary antibody-HRP polymer
primary antibody-HRP polymer/tyramide-peptide bridging antigen.
[0300] 1. First-round of binding of tyramide-peptide bridging
antigen as listed in Protocol G was performed. [0301] 2. To the
slide was added the anti-PEP6-HRP at 10 ng/.mu.L for 10 minutes.
[0302] 3. Repeat Protocol G steps 4-6 using a second round of
Tyr-PEP6. [0303] 4. To the slide was added the anti-PEP6-Dy650
(Dyomics GmbH, Jena, Germany) at 5 ng/.mu.L for 10 minutes. [0304]
5. Slides were washed 3.times. in wash buffer for 5 minutes each.
[0305] 6. Slides were rinsed with distilled water, removing excess
water with paper towel. [0306] 7. 1-3 drops of Fluoroshield with
DAPI (Immunobiosciences, Inc, cat# AR-6501-01) was added to each
slide and after 3-5 min in the dark at room temperature the
coverslip was applied.
[0307] Protocol L: Anti-rabbit secondary antibody-HRP-tyrosine
peptide conjugation protocol: The following protocol was used to
conjugate peptide AOA-YRYPYRY-NH.sub.2 (SEQ ID NO.:6) ("AOA-Ty4Pep"
to anti-rabbit HRP Polymer. Similar protocols were used to
conjugate the other tyrosine peptides to their respective secondary
HRP polymers. To a solution of anti-rabbit-HRP (58 uL; 150 .mu.g at
2.6 mg/mL; 0.455 nmol) in Modification Buffer was added a solution
of sulfo-4FB (0.64 .mu.L of a 2.5 mg/mL solution in DMSO; 4.6 nmol;
10 mol equiv; Cell IDx, San Diego, Calif.). The reaction was
incubated at room temperature for 2 h and desalted into Conjugation
Buffer using a 0.5 mL Zeba column pre-equilibrated with Conjugation
Buffer. The antibody recovery was assumed to be 90% (135 .mu.g)
based on previous Zeba column recovery rates. AOA-Tyr4Pep (0.71
.mu.L of a 5 mg/mL solution in DMF; 3.1 nmol: 15 mol equiv;
Innopep, San Diego, Calif.) was added to anti-rabbit-4FB, followed
by addition of aniline buffer (3.1 .mu.L) and incubated at room
temperature for 2 hours. Free peptide and aniline was removed by
using a Spin-X UF 30K molecular weight cutoff concentrator
(Corning, UK) by adding 3 separate additions of 10 mM phosphate,
150 mM NaCl, pH 7.0 buffer, of at least 5 fold the amount of sample
volume in the concentrator to ensure complete removal and buffer
exchange. The anti-rabbit HRP polymer-Tyr4Pep conjugate was spun
down at 10K rpm for 5 minutes to remove large aggregates. The
concentration of the antibody-peptide product was determined
spectrophotometrically using antibody extinction coefficient of 1.4
and a correction factor of 0.45.
Results
[0308] FIGS. 9A-9E show exemplary staining of Ki-67 on
triple-positive breast cancer tissues using different
tyramide-containing reagents. An unlabeled mouse anti-Ki-67 primary
antibody (at a concentration of 0.5 .mu.g/mL in each case) was
reacted with an anti-mouse secondary antibody labeled with
horseradish peroxidase (HRP). The samples were then treated with a
tyramide-labeled reagent compound and hydrogen peroxide, and in
some cases the samples were subsequently stained with a fluorescent
streptavidin or a fluorescent antibody specific for the
tyramide-labeled reagent. FIG. 9A:
tyramide-biotin/streptavidin-Dy650; FIG. 9B: tyramide-Dy650; FIG.
9C: tyramide-digoxigenin/anti-digoxigenin-Dy650; FIG. 9D:
tyramide-PEP1/anti-PEP1-Dy650; and FIG. 9E:
tyramide-PEP6/anti-PEP6-Dy650. Signals were normalized to the
output from the tyramide-biotin sample.
[0309] FIGS. 10A and 10B show exemplary staining of Ki-67 on
triple-positive breast cancer tissues using an anti-Ki-67 antibody
labeled with the PEP6 bridging antigen at 10 pg/.mu.L. The samples
were further treated with an anti-PEP6-HRP antibody reagent
followed by a tyramide-PEP6 reagent compound. A one-round staining
is shown in FIG. 10A, where the PEP6 antigen was detected using
anti-PEP6-Alexa567. A two-round staining is shown in FIG. 10B,
where the sample was further reacted with a second round of the
anti-PEP6-HRP antibody reagent and the tyramide-PEP6 reagent
compound to amplify the PEP6 label. The PEP6 antigen was then
detected using anti-PEP6-Alexa567 as in the one-round
procedure.
[0310] FIGS. 11A-11D show exemplary staining of Ki-67 on
triple-positive breast cancer tissues using an unlabeled anti-Ki-67
primary antibody at 100 pg/.mu.L followed by treatment with an
HRP-labeled anti-mouse secondary antibody. The images compare one
round of staining with tyramide-biotin/streptavidin-650 (FIG. 11A)
to one round of staining with tyramide-PEP6/anti-PEP6-Dy650 (FIG.
11B) and two rounds of tyramide-biotin/streptavidin-Dy650 (FIG.
11C) to two rounds tyramide-PEP6/anti-PEP6-Dy650 (FIG. 11D). The
background signal increases significantly in the two-round
amplification with tyramide-biotin (FIG. 11C vs. FIG. 11A).
[0311] FIGS. 12A-12D are similar to FIGS. 11A-11D, except that a
lower concentration of primary antibody was used. Specifically,
these slides show staining of Ki-67 on triple-positive breast
cancer tissues using an anti-Ki-67 antibody at 10 pg/.mu.L and an
HRP-labeled anti-mouse secondary antibody. The images compare one
round of staining with tyramide-biotin/streptavidin-650 (FIG. 12A)
to one round of staining with tyramide-PEP6/anti-PEP6-Dy650 (FIG.
12B) and two rounds tyramide-biotin/streptavidin-Dy650 (FIG. 12C)
to two rounds tyramide-PEP6/anti-PEP6-Dy650 (FIG. 12D). These
slides again illustrate the problematic background staining
typically seen in multi-round TSA straining with traditional
reagents (e.g., comparing FIG. 12A and FIG. 12C). In contrast,
there is little or no increase in background between the first and
second round staining with the instant reagent compounds (e.g.,
comparing FIGS. 12B and 12D).
[0312] FIGS. 13A-13H illustrate the staining of HER2 on
triple-positive breast cancer tissues using primary antibodies that
have been modified with a peptide bridging antigen. Specifically,
the anti-HER2 primary antibodies were modified with PEP5 and used
at decreasing concentrations to label tissue sections. After
labeling, the sections were reacted with one round or two rounds of
anti-PEP5-HRP and tyramide-PEP5 and then stained with
anti-PEP5-Dy650. FIG. 13A (one round) and FIG. 13B (two rounds) are
at 6.67 nM anti-HER2-PEP5; FIG. 13C (one round) and FIG. 13D (two
rounds) are at 667 pM anti-HER2-PEP5; FIG. 13E (one round) and FIG.
13F (two rounds) are at 67 pM anti-HER2-PEP5; and FIG. 13G (one
round) and FIG. 13H (two rounds) are at 6.67 pM anti-HER2-PEP5.
[0313] FIGS. 14A-14C show the results obtained from a system
corresponding to the scheme illustrated in FIG. 6, where a tyrosine
peptide was coupled to the antibody reagent. In particular, a
linkable tyrosine peptide, AOA-YRYPYRY-NH.sub.2, was prepared by
solid phase peptide synthesis and linked to 4FB-modified
anti-mouse-HRP secondary antibody polymer at two levels of
modification. The immunofluorescence staining of these two
constructs after reaction with a fluorescent tyramide were then
compared to an anti-mouse-HRP secondary antibody polymer lacking
the tyrosine peptide.
[0314] The three constructs were used to stain the estrogen
receptor (ER) on triple-positive breast cancer tissue. FIG. 14A:
anti-rabbit-HRP @ 0.1 ng/.mu.L/tyramide-Alexa567; FIG. 14B:
anti-rabbit-HRP-(tyrosine peptide)Low @ 0.1
ng/.mu.L/tyramide-Alexa567; and FIG. 14C: anti-rabbit-HRP-(tyrosine
peptide).sub.HIGH @ 0.1 ng/.mu.L/tyramide-Alexa567. As is apparent
from a comparison of these results, the staining observed in
samples labeled with the anti-rabbit-HRP constructs containing the
tyrosine peptide were significantly stronger than the staining
observed in the sample labeled with the unmodified anti-rabbit-HRP
polymer. Image analysis by Image J software demonstrates a 3-4 fold
higher signal in the section of FIG. 14C compared to that of FIG.
14A.
[0315] FIGS. 15A and 15B illustrate the use of a tandem-repeat
bridging antigen to increase staining in tissue sections. As shown
in these slides, the HER2 biomarker was labeled on triple-positive
breast cancer tissue using a rabbit anti-HER2 primary antibody at 5
pg/.mu.L and an HRP-labeled anti-rabbit secondary antibody. The
label was amplified using either a tyramide-PEP5 reagent compound
(FIG. 15A) or a tyramide-PEP5-3.times. tandem repeat reagent
compound (FIG. 15B). The slides were then labeled with an
anti-PEP5-antibody labeled with Dy490. After imaging, the signals
were normalized to the output from the tyramide-PEP5 sample. As is
clear in the comparison, the sample labeled with the 3.times.
tandem repeat bridging antigen generates a significantly stronger
signal in this assay than the sample labeled with a reagent
compound containing a bridging antigen comprising a single antigen
determinant.
[0316] All patents, patent publications, and other published
references mentioned herein are hereby incorporated by reference in
their entireties as if each had been individually and specifically
incorporated by reference herein.
[0317] While specific examples have been provided, the above
description is illustrative and not restrictive. Any one or more of
the features of the previously described embodiments can be
combined in any manner with one or more features of any other
embodiments in the present invention. Furthermore, many variations
of the invention will become apparent to those skilled in the art
upon review of the specification. The scope of the invention
should, therefore, be determined by reference to the appended
claims, along with their full scope of equivalents.
* * * * *
References